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Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Open  Knowledge  Commons  (for  the  Medical  Heritage  Library  project) 


http://www.archive.org/details/textbookofphysio1901simo 


A  TEXT-BOOK 


Physiological  Chemistry 


FOR 


STUDENTS  OF  MEDICINE  AND  PHYSICIANS. 


BY 


CHARLES  E.  SIMON,  M.D., 


Of  Baltimore,  Md. 


LEA    BROTHERS  &  CO., 

1MI  I  I.  \  LI.  I.I'll  I  A    A  N  I)    N  EW    YORK. 
I  901 . 


Entered  according  to  Act  of  Congress,  in  the  year  1901,  by 

LEA   BROTHERS  &  CO., 

In  the  Office  of  the  Librarian  of  Congress,  at  Washington.    All  rights  reserved. 


WESTOOTT    &    THOMSON, 
ELECTROTYPERS,    PHILADA. 


PRESS    OF 
WILLIAM    J.    DORNAN.    PHILADA. 


TO  MY   UNCLE, 
MR.    HENRY    T.    SIMON, 

THIS    VOLUME 

IS    AFFECTIONATELY     DEDICATED 

BY 

THE  AUTHOR. 


PREFACE. 


In  preparing  the  present  volume  on  Physiological  Chemistry  I 
have  endeavored  to  adapt  the  book  as  much  as  possible  to  the  wants 
of  the  medical  student,  and  the  physician  who  in  the  past  has  been 
unable  to  devote  the  attention  to  the  subject  which  it  merits.  The 
work  is  intended  as  a  text-book  for  the  lecture-room  and  as  a  guide 
in  the  physiological-chemical  laboratory.  Theoretical  discussions 
have  been  avoided  as  far  as  possible,  and  it  has  been  my  aim  to 
present  ascertained  facts  as  concisely  as  appeared  consistent  with  the 
importance  of  the  problems  under  consideration.  The  various 
chemical  methods  have  been  described  with  all  due  regard  to 
necessary  detail,  but  with  the  supposition  that  the  student's  course 
in  physiological  chemistry  has  been  preceded  by  a  course  in  general 
chemistry,  such  as  is  offered  now  in  the  majority  of  our  medical 
colleges. 

The  subject-matter  has  been  arranged  in  such  a  manner  that  in 
the  first  section  of  the  work  a  general  survey  is  given  of  the  origin 
and  the  chemical  nature  of  the  three  great  classes  of  food-stuffs,  and 
also  of  the  most  important  products  of  their  decomposition;  the 
second  section  deals  essentially  with  the  processes  of  digestion,  re- 
sorption, and  excretion  ;  while  the  third  portion  of  the  work  is 
devoted  to  the  chemical  study  of  the  elementary  tissues  and  the 
various  organs  of  the  animal  body,  the  specific  products  of  their 
activity  and  their  relation  to  physiological  function.  This  arrange- 
ment has  suggested  itself  to  me  as  the  most  satisfactory  for  purposes 
of  teaching. 

References  to  literature  have  been  omitted, as  they  did  not  appear 
t<»  Ik-  necessary  in  a  work  which  is  intended  primarily  for  the 
student.     The  names  of  tin-  grand-masters  of  physiological  chem- 


vi  PREFACE. 

istry  and  physiology,  however,  have  been  introduced  into  the  text 
as  a  matter  of  historical  interest. 

To  my  friend,  Mr.  Charles  Glaser,  of  Baltimore,  I  wish  to 
express  my  sincere  thanks  for  many  valuable  suggestions  and  aid 
in  the  revision  of  the  manuscript.  To  Messrs.  Lea  Bros.  &  Co. 
I  am  indebted  for  many  acts  of  courtesy. 

1302  Madison  Avenue, 
Baltimore,  Md.,  1901. 


CONTENTS. 


CHAPTER   I. 
INTRODUCTION. 

PAGE 

General  chemical  composition  of  living  matter 17 

Forces  at  work  in  the  living  world 17 

Character  of  chemical  changes      18 

Synthetic  processes  in  plants 18 

Oxidations  and  hydrations  in  the  animal  body 19 

Chlorophyl 21 

Chemical  nature  of  chlorophyl 21 

The  food-stuffs  of  the  plant 23 

Synthesis  of  the  carbohydrates 24 

Glucosides 25 

Mannides 26 

Synthesis  of  the  fats 26 

Synthesis  of  the  albumins 26 

CHAPTER  II. 

THE   ALBUMINS. 

Elementary  composition 30 

Crystallization 30 

Solubility 31 

Behavior  toward  neutral  salts  and  alcohol 31 

Diffusion 31 

Coagulation 32 

Denaturization 33 

Behavior  toward  polarized  light 33 

•  lolor-reactiooB 34 

The  xanthoproteic  reaction 34 

Millon's  reaction 34 

The  reaction  of  Adamkiewicz 34 

The  biuret  reaction 34 

Boiling  witli  hydrochloric  acid^furfurol  reaction 35 

Sulphur  t<st 35 

Molisch's  test 35 

Precipitation  of  1 1 » t -  albumins 36 

Decomposition  of  tin-  albumins 37 

Synthesis  of  the  albumins 38 

Molecular  size  of  the  albumins 38 

Structural  composition  of  the  albumins 39 

Classification  of  the  albumins 40 

vii 


viii  CONTENTS. 

PAGE 

The  Native  Albumins .  41 

The  albumins 41 

The  globulins 41 

The  vitellins 42 

The  Proteids 42 

Thenucleins 42 

The  nucleo-albumins 43 

The  glucoproteids 44 

The  mucins 45 

The  mucoids  or  ruucinoids 45 

The  hyalogens ,.  45 

The  haemoglobins 46 

The  Albuminoids 46 

The  keratins 47 

Elastin 47 

Collagen 47 

Glutin  (gelatin) 47 

The  skeletins 48 

Amyloid 48 

The  Derived  Albumins 48 

Fibrin 48 

The  coagulated  albumins 49 

The  albuminates 49 

The  albumoses 49 

The  peptones 51 

CHAPTER  III. 
THE   CAEBOHYDEATES. 

The  Monosaccharides 54 

Lsevulose 57 

Galactose 57 

Glucose 57 

The  Disaccharides 57 

Cane-sugar 58 

Maltose 59 

Isomaltose .    .    .  59 


Lactose 


59 


The  Polysaccharides 59 

Starch 60 

Inulin  and  lichenin bJ^ 


Glycogen 


61 


Dextrins 

Celluloses 61 

CHAPTER   IV. 

THE  FATS. 

The  Fats 63 

The  Lecithins °5 

The  Cholesterins 67 


CONTENTS.  ix 

CHAPTER   V. 

THE   NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

PAGE 

The  Protamins 69 

The  Protons 71 

The  Hexon  Bases 71 

Arginin 72 

Lysin 73 

Histidin 74 

The  Ntjcleimtc  Acids 74 

The  Xucleixic  Bases 77 

The  Ureids 81 

Uric  acid 82 

The  Kreatins 84 

The  Amido-acids 85 

The  Ptomains 91 

The  Organic  Non-nitrogenous  Acids 93 

CHAPTER  VI. 

THE  FERMENTS. 

General  properties 101 

Chemical  composition  and  general  reactions 103 

Mode  of  action 104 

Classification 104 

The  proteolytic  ferments 105 

The  amylolytic  ferments 105 

The  inverting  ferments 105 

The  steatolytic  ferments 105 

The  coagulating  ferments 105 

The  ferments  which  decompose  urea 105 

The  ferments  which  decompose  the  glucosides 105 

The  tissue- ferments 106 


CHAPTER  VII. 
THE   DIGESTIVE  FLUIDS. 

The  Saliva l°8 

General  characteristics 108 

Amount 108 

Chemical  composition 109 

I'tyalin HO 

Mucin 11- 

Sulphocyanides ''•'* 

Nitrites 113 

Extractives * 

Mineral  Constituents H"* 

M ^'4 


x  CONTENTS. 

PAGE 

The  Gastric  Juice • ,  114 

General  considerations 114 

Amount 114 

Chemical  composition 115 

Acidity  c-f  the  gastric  juice 115 

Determination  of  the  total  acidity  of  the  gastric  contents 116 

Amount 117 

Hydrochloric  acid 117 

Origin 117 

Significance 118 

Tests  for  hydrochloric  acid 119 

Estimation 120 

Lactic  acid 122 

Tests 122, 

Estimation 122 

Acetic  acid  and  butyric  acid 123 

Tests 123 

Estimation 123 

Quantitative  estimation  of  the  organic  acids 124 

The  ferments  of  the  gastric  juice  and  their  pro-enzymes 124 

Pepsin 126 

Isolation 127 

Estimation 129 

Pepsinogen 129 

Tests 129 

Estimation 130 

Chymosin  and  chymosinogen 130 

Tests 131 

Isolation 131 

Estimation 132 

Other  constituents  of  the  gastric  juice 132 

Gases 132 

The  Pancreatic  Juice 133 

General  properties 134 

Specific  gravity .  134 

Amount 134 

Chemical  composition 134 

The  ferments  and  their  zymogens 136 

Trypsin 136 

Test 137 

Isolation 138 

The  amylolytic  ferment 138 

Steapsin 138 

Maltase 139 

Chymosin 139 

The  glucolytic  ferment 139 

The  Enteric  Juice 140 

TheBiee 141 

Secretion 142 

Amount 143 


CONTENTS.  xi 

PAGE 

General  properties 143 

Chemical  composition 144 

The  mucinous  body  of  the  bile 145 

The  biliary  acids 145 

Isolation 147 

Tests 147 

Pettenkofer's  test 147 

Physiological  test 148 

Glycocholic  acid 148 

Hyoglycocholie  acid 149 

Taurocholic  acid 149 

Hyotaurocholic  acid 150 

Chenotaurocholic  acid 150 

Cholalic  acid 151 

Hyocholalic  acid  and  chenocholalic  acid 153 

Choleic  acid 153 

Fellic  acid 153 

Lithofellic  acid 153 

Taurin 153 

Isolation 154 

Glycocoll 155 

The  bile-pigments 155 

Bilirubin 156 

Tests 158 

Isolation 159 

Biliverdin 160 

Isolation 160 

Biliprasin 161 

Bilifuscin 161 

Bilicyanin 161 

Bilipurpurin 161 

Choletelin 161 

Bilihumin 162 

Cholesterin 162 

Tests 163 

Other  organic  constituents  of  the  bile 163 

Tbe  biliary  iron 164 


CHAPTER  VIII. 

THE    PROCESSES   OF   DIGESTION  AND  RESORPTION. 

The  Digestion  am.  Resorption  of  the  Cabbohydbates 165 

The  Digestioh  am.  Resorption  op  the  AuiniuiNs 168 

Digestion  of  the  native  albumins 168 

Gastric  digestion 168 

Tryptic  digestion ''' 

Digestion  of  the  proteids  .   .              1,s 

Digestion  of  the  albuminoids ''■' 

The  Digestion  am.  Resorption  op  the  Fats 180 


xn  CONTENTS. 

CHAPTER   IX. 
ANALYSIS  OF  THE  PEODUCTS  OF  ALBUMINOUS  DIGESTION. 

PAGE 

The  Products  of  Peptic  Digestion 183 

The  albumoses 183 

The  Products  of  Tryptic  Digestion 185 

Leucin 185 

Tyrosin 185 

Asparaginic  acid 190 

Glutaruinic  acid 191 

Glycocoll 192 

Tryptophan 193 

Antipeptone 195 

CHAPTER   X. 

BACTEEIAL  ACTION  IN  THE  INTESTINAL  TEACT. 

Indol 198 

Skatol 199 

Phenol 200 

Ptomains 201 

Bacterial  Decomposition  of  the  Fats 202 

Bacterial  Decomposition  of  the  Biliary  Constituents 202 

CHAPTER   XI. 

THE  FECES. 

Consistence  and  form 204 

Amount 204 

Odor 204 

Color 204 

Macroscopical  constituents 205 

Microscopical  constituents 205 

Reactions 205 

General  chemical  composition 205 

Analysis  of  the  Products  of  Albuminous  Putrefaction 206 

Hydrobilirubin 207 

Excretin 208 

Stercorin 208 

.Meconium 208 

CHAPTER  XII. 

THE  UEINE. 

General  Characteristics 209 

General  appearance 209 

Color 211 

Odor 211 


CONTENTS.  xiii 

PAGE 

Amount 211 

Specific  gravity 212 

Keaction 212 

Determination  of  the  acidity  of  the  urine 215 

Chemical  composition 216 

The  Inorganic  Constituents  of  the  Urine 216 

Quantitative  estimation  of  the  mineral  ash 219 

Quantitative  estimation  of  the  chlorides 219 

Quantitative  estimation  of  the  phosphates 220 

Separate  estimation  of  the  earthy  and  alkaline  phosphates 220 

Quantitative  estimation  of  the  sulphates 220 

Test  for  nitrates 221 

The  Organic  Constituents  of  the  Urine 221 

The  nitrogenous  constituents  of  the  urine 222 

Urea 222 

Origin 222 

Nitrogenous  equilibrium 225 

Properties 227 

Urea-nitrate 227 

Urea-oxalate 228 

Synthetic  formation 229 

Isolation 230 

Quantitative  estimation 230 

Estimation  of  the  preformed  ammonia 232 

Estimation  of  the  total  urinary  nitrogen 232 

Uric  acid 233 

Origin 233 

Properties 236 

Tests 237 

Isolation 237 

Quantitative  estimation 238 

The  xanthin-bases 240 

Origin 240 

Quantitative  estimation 241 

Oxalic  acid  and  oxaluric  acid 241 

Quantitative  estimation  of  oxalic  acid 243 

Allantoin 243 

Isolation 244 

Kreatinin 241 

Properties 245 

Tests 240 

Synthesis 246 

Isolation  and  quantitative  estimation 246 

Tin;   Lbomatic  Constituents  ov  the  1'kini: 247 

The  conjugate  sulphates 248 

The  phenols 248 

Quantitative  estimation 249 

[ndoxyl  sulphate 250 

252 

Quantitative  estimation 253 


xiv  CONTENTS. 

PAGE 

Skatoxyl  sulphate 253 

Tests 254 

The  conjugate  glucuronates 255 

The  compound  glycocolls 256 

Hippuric  acid 257 

Properties 257 

Synthesis 258 

Isolation 258 

Quantitative  estimation 259 

Phenaceturic  acid 259 

Properties 259 

Isolation 259 

Ornithuric  acid 260 

The  Aromatic  Oxy-acids 260 

Homogentisinic  acid 261 

Inosit 262 

Kynurenic  acid 263 

The  Fatty  Acids 264 

The  volatile  fatty  acids 264 

Isolation  and  quantitative  estimation 264 

/?-oxybutyric  acid 265 

Test 265 

Diacetic  acid 266 

Tests 266 

Acetone 267 

Tests 267 

Quantitative  estimation 268 

Ehrlich's  reaction 268 

Paralactic  acid 269 

Isolation 269 

Leucin  and  tyrosin 270 

Isolation 271 

The  Neutral  Sulphur  Bodies  of  the  Urine 271 

Cyste'm .  272 

Cystin 273 

Quantitative  estimation  of  the  neutral  sulphur 275 

The  Carbohydrates 275 

Glucose 276 

Tests 278 

Quantitative  estimation 280 

Lactose 282 

Isolation 282 

Leevulose 283 

Laiose 283 

Maltose 283 

Dextrin 283 

Pentoses 283 

The  Albumins 284 

Tests ' 286 

Estimation 290 


CONTENTS.  xv 

PAGE 

The  Pigments  of  the  Urine 291 

Crochrome 291 

Isolation 292 

Uroerythrin 292 

Isolation 293 

Urobilin 293 

Tests 293 

The  blood- pigments 294 

Hsematin 295 

Haematoporphyrin 295 

Urorubrohsernatin  and  urofuscohavmatin 296 

Melanins 296 

The  bile-pigments 297 

The  bile-acids  ■. 297 

Fats,  cholesterin,  and  lecithins 298 

Ferments 298 

Oases 299 

Ptomains 299 


CHAPTER   XIII. 

THE  ANIMAL  CELL. 

Protoplasm  and  nucleus 301 

Chemical  changes 302 

CHAPTER   XIV. 

THE   BLOOD. 

General  considerations 306 

Physical  Characteristics  op  the  Blood 307 

Color 307 

Odor 308 

Taste 308 

Specific  gravity 308 

Amount 309 

Chemical  Examination  of  the  Blood 309 

Reaction 309 

Chemical  composition  of  the  blood  as  a  whole 310 

The  plasma 312 

Fibrinogen 313 

[eolation 313 

Properties 314 

Sernm-globulin 314 

Isolation 314 

Properties 315 

Serum-albumin 316 

Separation  of  the  albumins  from  each  other 316 

Quantitative  estimation  of  the  albumins 316 


xvi  CONTENTS. 

PAGE. 

Serum 317 

The  fibrin  ferment 319 

Isolation  .    .        319 

Properties 319 

Fibrin 319 

Estimation 321 

Glycogen 321 

Fat 322 

Urea , 322 

The  leucocytes 322 

Nucleohiston • 322 

Isolation 322 

Properties 323 

Chemical  composition * 323 

The  plaques 324 

The  coagulation  of  the  blood 324 

Rapidity  of  coagulation 326 

The  red  corpuscles 327 

Isolation 328 

Haemoglobin  and  its  derivatives 328 

Haemoglobin 328 

Globin 330 

Hsemochromogen 330 

Oxyhaemoglobin 331 

Haematin 331 

Carbon  dioxide  haemoglobin 335 

Carbon  monoxide  haemoglobin 335 

Nitric  oxide  haemoglobin 336 

Methsemoglobin 336 

Haematoporphyrin 337 

Phylloporphyrin 338 

Haematoidin 338 

Haemocyanin 338 


CHAPTER  XV. 

THE   LYMPH. 

Chemical  composition 341 

Analyses  of  different  forms 343 

Analysis  of  pericardial  fluid 343 

Analysis  of  chyle 343 

Analysis  of  cerebrospinal  fluid 344 

Analysis  of  pleural  effusion 344 

Analysis  of  peritoneal  effusion 344 

Analysis  of  hydrocele  fluid 344 

Analysis  of  amniotic  fluid 344 

Analysis  of  lymph 344 

Analysis  of  pus 345 

The  Synovial  Fluid 345 


CONTENTS.  xv  11 

CHAPTER   XVI. 
THE   MUSCLE-TISSUE. 

PAGE 

Analysis  of  fresh  muscle- tissue 346 

The  Muscle-albumins 347 

Muscle-plasma 347 

Myogen 347 

Isolation 34S 

Myosin 349 

Significance  of  the  common  muscle-albumins 350 

Other  albumins 351 

Myoproteid 351 

Nucleus 351 

Phosphor-carnic  acid 351 

Ferments 352 

Muscle-stroma 352 

The  Muscle-pigments 353 

Glycogen 353 

Glucose 356 

Lactic  Acid 356 

Isolation  and  quantitative  estimation 358 

[hosit 359 

Tests 360 

Isolation 360 

The  Nitrogenous  Extractives 360 

Kreatin  and  kreatinin 360 

Properties  of  kreatin 362 

Isolation  of  kreatin 362 

The  xantliin-bases 363 

Isolation 363 

Xantliin 364 

Iivpoxanthin 365 

Guanin 365 

Adenin 366 

Carnirj 366 

Inosinic  acid 367 

1 1  uses 367 

Fat 36S 

CHAPTER   XVII. 

THE    NERVE-TISSUE. 

Analysis 370 

Albumin- 370 

Neurokeratin •"" ' 

lin  Mvi:i.in  Bodies •"•""-' 

Protagon 3,2 

brin 373 

I  [omocerebrin 374 


xv  in  CONTENTS. 

PAGE 

Encephalin 375 

Lecithins 375 

Cholesterins 376 

The  extractives , 376 

Neuridin 376 

Jecorin 376 

CHAPTER   XVIII. 

THE   EYE  AND  THE   EAE. 

The  Eye 377 

The  cornea 377 

The  sclerotic 377 

The  aqueous  humor 377 

The  crystalline  lens 377 

The  vitreous  body 379 

The  retina 379 

Rhodopsin 380 

Chromophanes 381 

The  choroid 381 

The  Ear 381 

CHAPTER  XIX. 

THE  SUPPOKTING  TISSUES. 

Mucous  tissue 382 

White  fibrous  tissue 382 

Yellow  elastic  tissue 383 

Reticulated  tissue 383 

Cartilage 383 

Chondroitin  sulphuric  acid 384 

Chondromucoid ' 385 

Albumoid • 385 

Mineral  constituents 386 

Bone 386 

Bone-marrow 388 

The  Teeth 388 

Dentin 388 

Cement 388 

Enamel 388 

Adipose  Tissue 389 

Analysis  of  adipose  tissue 390 

Origin  of  the  fats 391 

Significance  of  the  fats 393 

CHAPTER   XX. 
THE   SKIN  AND  ITS  APPENDAGES. 

The  sweat 395 

Gases 397 

The  sebum 397 

The  cerumen SOS 


CONTENTS.  xix 

CHAPTER  XXI. 

THE  GLANDULAR  OEGANS  OF  THE  BODY. 

PACE 

The  Liver 399 

The  albumins 400 

Ferments 40"J 

Glycogen 403 

Glucose 404 

Fat .  404 

Extractives 404 

The  Digestive  Glands 405 

The  Lymph-glands 405 

The  Kidneys 406 

The  Mammary  Glands ...  406 

The  milk .  407 

General  characteristics 407 

Amount 409 

Specific  gravity „ 409 

Reaction 410 

Chemical  composition .410 

The  albumins 4H 

Casein 411 

Lactalbumin 413 

Lactoglobulin 414 

The  fats 41.^ 

Lactose . 410 

The  extractives 417 

Colostrum 417 

The  Reproductive  Glands  .              .       41!) 

The  testicles 4ID 

The  semen 41<) 

The  spermatic  liquid.       420 

Spermin 4->() 

The  spermatozoa .  421 

The  ovaries 4>22 

The  ovum        4^2 

The  yolk  .    .                   |.j(; 

Incubation aoq 

CHAPTER   XXII. 

THE   DUCTLE8S  GLANDS. 

The  Thyroid  Gland 439 

Thyreoglobulin          433 

Thyreo-niicleo-albumin         434 

Extractives  ...           435 

Tin.  Adrenal  Glands i.;, 


PHYSIOLOGICAL    CHEMISTRY. 


CHAPTER    I. 

INTRODUCTION. 


The  science  of  physiological  chemistry  has  for  its  object  the 
study  of  the  various  chemical  processes  which  take  place  in  the 
bodies  of  animals  and  plants,  and  which  are  more  or  less  intimately 
associated  with  the  phenomena  of  life.  As  the  phenomena  of  life, 
moreover,  are  essentially  dependent  upon  the  transformation  of 
living  matter  into  non-living  matter,  and  vice  versa,  physiological 
chemistry  deals  primarily  with  the  chemical  processes  of  nutrition 
in  the  widest  sense  of  the  term.  Its  study  therefore  comprises  a 
consideration  of  the  various  substances  which  are  generally  desig- 
nated as  food-stuffs,  their  origin,  their  transformation  into  living 
tissue,  and  their  ultimate  fate. 

General  Composition  of  Living  Matter. — Chemical  examina- 
tion shows  that  plants  and  animals  consist  essentially  of  carbon, 
hydrogen,  nitrogen,  oxygen,  sulphur,  phosphorus,  chlorine,  potas- 
sium, sodium,  calcium,  magnesium,  and  iron — that  is,  of  elements 
which  occur  also  widely  distributed  in  the  non-organized  world. 
In  the  bodies  of  animals  and  plants  these  elements  are  built  up  to 
form  bodies  of  highly  complex  chemical  constitution,  which  belong 
to  the  class  of  albumins,  carbohydrates,  and  fats.  Upon  their  pres- 
ence both  animals  and  plants  are  dependent  for  their  existence,  and 
as  these  bodies  are  constantly  being  broken  down  and  transformed 
into  Bimpler  chemical  compounds,  as  the  result  of  the  various  mani- 
festations  of  lite,  it  follows,  from  the  law  of  the  indestructibility 
of  matter,  that  for  their  replacement  the  living  body  is  forced  to 
depend  upon  such  simpler  matter  as  is  pre-e.\istent.  I  nis  matter 
it  is  capable  of  transforming  into  the  complex  substances  of  which 
it  -  tissues  are  composed. 

Forces  at  Work  in  the  Living-  World. — The  forces  which  are 
at  work  in  effecting  these  various  changes  are  the  same  as  those 
which  we  meet  with  in  the  non-organized  world.  They  represent 
essentially  n  transformation  of  energy,  of  which,  as  we  shall  presently 
Bee,  the  -nnlight  is  the  ultimate  source.  Ii  would  thus  appear  that 
life  iii  itself  i-  solely  a  physical  phenomenon,  and  that  its  various 

2  17 


1 8  INTR  OD  UCTION. 

manifestations  can  be  reduced  to  simple  physical  laws.  This  state- 
ment, however,  is  true  only  in  part,  for  although  the  forces  of  which 
we  have  cognizance  as  being  at  work  in  living  bodies  are  the  same 
as  those  with  which  we  are  familiar  in  the  non-organized  world, 
we  are  nevertheless  unable  to  explain  the  phenomena  of  life  upon 
this  basis,  and  are  as  yet  forced  to  accept  the  existence  of  a  vital 
principle,  of  the  nature  of  which  we  know  nothing. 

Character  of  Chemical  Changes. — The  chemical  processes 
which  are  involved  in  the  transformation  of  non-living  matter  into 
living  tissue  are  qualitatively  the  same  in  plants  and  animals. 
Quantitative  differences,  however,  exist,  which  are  sufficiently  pro- 
nounced to  serve  as  marks  of  distinction  between  animals  and 
plants.  Thus  plants  are  capable  of  evolving  from  relatively  simple 
compounds  those  complex  chemical  substances  which  go  to  form 
their  structure,  while  animals  apparently  do  not  possess  this  power. 
They  are  hence  dependent  for  their  existence  upon  food-stuffs  which 
are  preformed ;  and  the  potential  energy  which  animals  require 
for  the  functioning  of  their  various  organs,  and  which  they  trans- 
form into  kinetic  energy,  is,  as  a  matter  of  fact,  derived  in  every 
instance,  either  directly  or  indirectly,  from  plant-life.  Plants,  in 
turn,  obtain  the  potential  energy  which  is  stored  in  their  tissues  from 
the  kinetic  energy  of  sunlight,  and  in  virtue  of  this  energy  can 
elaborate  those  simple  chemical  substances  which  are  at  their  dis- 
posal as  food-stuffs  into  the  complex  bodies  which  constitute  their 
tissues. 

We  thus  observe  that  while  in  plant-life  synthetic  chemical 
processes  prevail,  analytical  processes  are  foremost  in  animal  life. 
These  analytical  processes,  moreover,  are  largely  of  the  character  of 
oxidations,  while  the  syntheses  which  are  effected  in  the  bodies  of 
plants  are  essentially  of  the  nature  of  reductions.  But  just  as  syn- 
thetic processes  are  not  absolutely  characteristic  of  plant-life,  so  also 
do  oxidation-processes  occur  in  plants,  and  synthetic  reductions  in 
animals.  This  becomes  especially  noticeable  as  we  descend  in  the 
scale  of  both  animal  and  vegetable  life.  Primitive  vegetable 
organisms  are  thus  met  with  which,  like  the  highly  organized  mam- 
mal, are  almost  entirely  dependent  for  their  existence  upon  already 
elaborated  food-stuffs,  and  low  forms  of  animal  life  similarly  occur 
in  which  the  processes  of  nutrition  are  essentially  the  same  as  those 
which  occur  in  the  higher  plants.  The  differences  which  thus  exist 
between  animal  life  and  plant-life  are  therefore,  as  has  been  stated, 
more  of  a  quantitative  than  a  qualitative  kind. 

Synthetic  Processes  in  Plants. — I  have  said  that  plants  are 
capable  of  elaborating  from  simpler  compounds  the  complex  chem- 
ical substances  of  which  they  are  composed,  and  that  the  chemical 
processes  here  involved  are  essentially  of  the  nature  of  synthetic 
reductions.  Formerly,  it  was  believed  that  the  various  organic  sub- 
stances which  occur  in  animals  and  plants  could  be  produced  only 
through  the  agency  of  a  special  vital  force ;  but  we  now  know  that 


OXIDATIONS  AND  HYDRATIONS  IN  THE  ANIMAL  BODY.    19 

this  is  not  necessarily  the  case,  and  that  as  a  matter  of  fact  a  large 
number  of  such  bodies  can  be  produced  artificially  in  the  chemical 
laboratory.  Wohler,  in  1829,  was  the  first  to  demonstrate  this 
possibility  by  preparing  urea  from  ammonium  cyanate.  This  he 
accomplished  by  heating  the  substance  to  a  temperature  of  100°  C, 
when  a  transposition  of  atoms  apparently  takes  place,  and  urea 
results.  The  force  which  is  necessary  to  effect  such  a  change  is 
here,  as  in  many  syntheses  which  can  artificially  be  brought  about, 
a  relatively  high  temperature.  In  the  bodies  of  animals  and  plants 
a  like  temperature,  of  course,  would  destroy  life,  and  there  must 
hence  be  a  different  mechanism  at  the  disposal  of  living  beings  to 
effect  such  a  change.  Of  the  nature  of  this  mechanism,  however, 
we  know  but  little,  and  we  are  forced  to  admit  that  while  it  is  pos- 
sible to  produce  chemical  substances,  such  as  those  which  are  found 
in  the  living  world,  by  artificial  means,  plants  and  animals  have 
manifestly  other  forces  at  their  command  which  are  more  or  less 
intimately  associated  with  that  peculiar  phenomenon  we  term  life. 
We  know  that  under  the  influence  of  sunlight  certain  plants  are 
capable  of  effecting  the  synthesis  of  carbohydrates,  fats,  and  albu- 
mins from  the  carbon  dioxide  of  the  air,  and  the  water  and  certain 
mineral  salts  of  the  soil,  and  that  the  ability  to  bring  about  these 
changes  is  in  a  large  measure  dependent  upon  the  presence  of  a 
chemical  substance  which  is  found  in  the  green  parts  of  plants,  and 
which  is  termed  chlorophyl.  We  know  further  that  chlorophyl 
requires  exposure  to  sunlight  to  effect  these  changes,  but  of  the 
mechanism  through  which  these  changes  are  brought  about  we  know 
nothing. 

Oxidations  and  Hydrations  in  the  Animal  Body. — The  oxida- 
tion-processes which  prevail  in  animals,  and  in  consequence  of  which 
the  more  complex  substances  which  go  to  form  the  various  tissues 
and  organs  of  the  body  are  retransformed  into  those  simple  com- 
pounds which  plant-  require  for  their  existence,  we  are  also  un- 
able to  explain  on  the  basis  of  simple  physical  laws.  We  know 
that  the  oxygen  of  the  air,  as  also  that  of  the  blood,  exists  in  a 
neutral  molecular  form,  and  is  as  such  incapable  of  effecting  the 
oxidation  of  such  complex  substances  as  the  albumins  and  fats. 
The  older  view  that  oxygen  exists  in  the  body  as  ozone,  and  that 
the  various  oxidation-processes  take  place  in  the  animal  fluids,  has 
been  abandoned,  and  it  is  now  generally  accepted  that  these  changes 
occur  iii  the  individual  cells.  Here,  then,  a  splitting  up  of  the 
neutral  oxygen  must  take  place,  but  of  the  forces  which  effect 
this  decomposition  we   know  next  to  nothing.     Whether  we  believe 

with   Pfluger  that   the  organized  living  albumin,  in    contradistinction 

to   the    non-organized    circulating   albumin,  i<   characterized    by  a 

greater    motility  of  it-   atom-,  iii    consequence  of  which    the   neutral 

oxygen  i-  decomposed,  or  whether  we  accept  the  view  that  reducing- 
-uli-ianee-  are  formed  during  the  decomposition  of  the  albuminous 
molecule  in   consequence  of  the  activity  of  a  third   factor,  we  are 


20  IX  TB  OD  UCTION. 

as  far  removed  from  an   adequate   explanation  of  these  phenomena 
as  in  the  beginning. 

Within  recent  years  repeated  observations  have  shown  that  from 
various  organs  of  the  body  certain  substances  can  be  extracted  which 
are  apparently  identical  with  or  closely  related  to  the  so-called  en- 
zymes. Certain  representatives  of  this  class,  such  as  pepsin,  trypsin, 
ptyalin,  and  others,  are,  as  we  shall  see,  formed  in  the  cells  of  the 
digestive  glands  of  the  body,  and  serve  the  purpose  of  transforming 
the  various  food-stuffs  which  are  furnished  the  animal  by  the  plant 
into  forms  which  can  be  absorbed  and  built  up  into  its  tissues.  The 
chemical  processes  which  are  here  involved  are  essentially  of  the 
character  of  hydrations.  Other  bodies,  however,  of  this  order 
which  can  be  obtained  from  living  tissues,  and  which  are  also 
capable  of  manifesting  their  special  activity  after  the  death  of  their 
parent-cells,  apparently  possess  the  power  of  oxidation,  and  it  is 
hence  supposed  by  some  that  these  processes  in  the  living  tissues 
may  also  be  referable  to  such  enzymotic  activity.  Whether  this 
is  actually  the  case  is  not  definitely  known.  But  if  so,  we  are 
apparently  approaching  a  time  when  what  we  have  heretofore  been 
forced  to  ascribe  to  the  activity  of  a  special  vital  force  may  be 
explained  upon  the  basis  of  physical  laws  which  are  seen  also  at  work 
in  the  non-organized  Avorld.  For  we  know  that  properties  which  are 
supposedly  characteristic  of  the  enzymes  are  possessed  also  by  cer- 
tain elements  which  are  found  only  in  the  inorganic  world.  The 
most  notable  properties  of  the  enzymes  are  their  ability  to  effect  an 
amount  of  chemical  change  which  is  out  of  all  proportion  to  the 
quantity  of  the  enzyme  present,  and  the  fact  that  the  enzyme  itself 
apparently  does  not  enter  into  the  reaction.  These  same  properties, 
however,  are  common  to  certain  metals  and  their  oxides..  Bredig 
and  von  Berneck  showed  that  a  gram-atomic  weight  (193  grams)  of 
colloidal  platinum  diffused  through  70,000,000  liters  of  water  shows 
a  perceptible  action  on  more  than  1,000,000  times  the  quantity  of 
hydrogen  peroxide  ;  and  H.  C.  Jones  demonstrated  that  the  reac- 
tion which  here  takes  place  is  a  mono-molecular  reaction,  which 
indicates  that  the  platinum  itself  does  not  enter  into  the  reac- 
tion. Curiously  enough,  the  analogy  between  the  action  of  such 
metallic  solutions  and  that  of  the  enzymes  goes  still  further.  Finely 
divided  platinum,  palladium,  iridium,  osmium,  etc.,  thus  have  the 
power  of  inverting  cane-sugar,  like  one  of  the  enzymes,  invertin  ; 
and  certain  poisons,  such  as  hydrocyanic  acid,  sulphuretted  hy- 
drogen, carbon  disulphide,  and  mercuric  chloride,  which  inhibit  or 
even  suspend  the  action  of  the  enzymes  entirely,  exert  a  similar 
influence  upon  a  solution  of  colloidal  platinum.  Without  entering 
upon  this  very  interesting  subject  further,  it  is  clear  that  a  path  has 
been  opened  upon  which  it  may  be  possible  to  penetrate  into  the 
mysteries  of  the  so-called  vital  forces,  and  to  show  ultimately  that 
such  forces  are  essentially  the  same  as  those  met  with  in  the  non- 
living world. 


CHLOROPHYL.  21 

Chlorophyl. — In  the  light  of  more  reeent  investigation,  it  seems 
probable  that  some  of  the  synthetic  processes  which  occur  in  plant- 
life  may  also  be  referable  to  the  action  of  enzymes.  As  a  matter 
of  fact,  such  bodies  are  abundantly  present  in  the  vegetable  world, 
and  we  know  that  some  of  these  at  least,  and  probably  all,  are 
characterized  by  a  reversible  activity.  Maltase,  a  ferment,  which, 
as  we  shall  see  later,  causes  inversion  of  the  disaccharide  maltose  to 
glucose,  is  thus  similarly  able  to  bring  about  the  synthetic  formation 
of  maltose  from  two  molecules  of  glucose.  On  the  other  hand,  it 
appears  that  the  primary  formation  of  food-stuffs  in  plants  does 
not  occur  in  this  manner,  but  is  referable  to  the  activity  of  a  special 
body  which,  as  has  been  stated,  is  present  in  the  exposed  green 
parts  of  most  plants,  and  which  is  termed  chlorophyl.  This  sub- 
stance occurs  widely  distributed  in  the  vegetable  world,  but  is  also 
found  in  those  low  forms  of  animal  life  in  which  the  processes  of 
nutrition  are  essentially  the  same  as  those  met  with  in  the  higher 
plants.  In  itself,  however,  chlorophyl  is  incapable  of  bringing 
about  those  syntheses  which  are  characteristic  of  vegetable  life,  and 
in  the  cells  of  the  foliage  of  plants  it  occurs  in  combination  with 
certain  albuminous  bodies,  in  the  form  of  the  so-called  chlorophvlic 
granules.  These  are  apparently  special  elementary  organisms,  and 
endowed  with  a  power  of  locomotion  analogous  to  that  of  amcebse 
and  leucocytes,  so  that  they  can  approach  the  surface  of  the  leaf  to 
seek  the  sunlight,  or  retreat  when  this  becomes  too  intense.  In  a 
germinating  plant  which  has  not  been  exposed  to  sunlight  the  green 
color  is  wanting,  but  in  the  cotyledons  is  found  a  differentiation  of 
the  cellular  protoplasm  into  small  yellowish  bodies,  which  have  been 
termed  leucltes.  When  these  bodies  are  exposed  to  light,  even  for  a 
relatively  short  time,  they  assume  a  green  color,  and  then  constitute 
the  chlorophvlic  grannies.  I  have  said  that  chlorophyl — that  is, 
the  green  coloring-matter  of  plants — is  unable  to  effect  synthetic 
changes,  and  the  same  is  true  of  the  colorless  leucites.  Chlo- 
rophyl thus  apparently  forms  an  integral  constituent  of  the  function- 
ing leucites,  and  all  those  chemical  and  physical  influences  which 
bring  about  the  destruction  of  the  protoplasm  similarly  destroy  the 
power  of  chlorophyl  to  manifest  its  special  activity.  In  the  living 
plant,  however,  it  becomes  active  at  once  upon  exposure  to  sunlight, 
and  i-  then  capable  of  effecting  those  complicated  syntheses  of  which 
mention  has  been  made.  In  the  dark  it  again  becomes  inactive,  and 
the  plant  i~  then  obliged  to  live  like  an  animal — by  consuming  its 
stored  energy. 

Chemical  Nature  of  Chlorophyl. — Of  the  chemical  composi- 
tion and  Structure  of  chlorophyl  we  know  little  that  is  definite. 
Numerous  attempts  have  been  made  to  isolate  it  from  the  living 
plant,  but  it  i-  doubtful    whether  any  of  these  attempts  has  yielded 

the  actual   substance.    Only  its  decomposition-prodilcts,  or  at  best 

very  impure    form-,    have  apparently  been    obtained,      (iautier,  it    is 

true,  claims   to  bave  isolated   the  substance  in  crystalline'fbrm  by 


22  INTRODUCTION. 

methods  which  are-  calculated  to  avoid  its  chemical  alteration. 
Others,  however,  have  not  been  successful  in  repeating  his  work. 

The  substance  which  Gautier  obtained  from  spinach-leaves  oc- 
curred in  the  form  of  small  crystals  of  a  dark-green  color,  which 
on  exposure  to  light  turned  brown,  then  yellow,  and  finally  became 
colorless.  Its  composition  corresponded  to  the  formula  C^H^NgC^. 
The  mineral  ash  consisted  of  about  1.75  per  cent,  of  magnesium 
phosphate,  traces  of  calcium  and  sulphates,  while  iron  was  absent. 
Treated  with  hydrochloric  acid,  it  was  decomposed  into  phylloxanthin 
and  phyllocyanic  acid,  C19H22N2303  or  C,8H20N2O3.  This  latter  is 
thus  a  homologue  of  bilirubin,  [C16H18N203]2,  which  in  turn  is 
derived  from  haematin,  and  is  isomeric  with  hcematoporphyrin.  A 
most  interesting  relationship  between  the  blood  coloring-matter 
haemoglobin  and  the  vegetable  coloring-matter  chlorophyl  thus 
becomes  apparent,  and  constitutes  a  further  link  connecting  the 
animal  with  the  vegetable  world.  Recent  investigations  have 
shown  that  a  substance  can  be  obtained  from  chlorophyl,  termed 
phylloporphyrin,  which  differs  only  from  hsematoporphyrin  anhy- 
dride in  containing  three  atoms  less  of  oxygen,  viz.,  C32H34N4(32. 
Both  bodies  are  thus  clearly  different  oxidation-products  of  one  and 
the  same  substance. 

Moderately  concentrated  solutions  of  chlorophyl  in  alcohol  or 
petroleum-ether  show  seven  bands  of  absorption.  The  first  of  these, 
I,  is  situated  in  the  red  portion  of  the  spectrum  between  B  and  C, 
and  is  well  pronounced  and  sharply  defined  on  both  sides.  The 
bands  II,  III,  and  IV  are  rather  indistinct  and  scattered  through 
the  orange-yellow,  the  yellow  and  the  yellowish-green  portion  be- 
tween C  and  E.  From  F  off,  the  greater  portion  of  the  spectrum 
is  absorbed  by  the  remaining  bands,  V,  VI,  and  VII,  of  which  V  is 
seen  to  the  right  of  F,  VI  most  marked  about  C,  while  VII  occu- 
pies the  extreme  violet  end.  Very  concentrated  solutions  allow  the 
red  rays  to  pass  only  as  far  as  B,  while  in  greater  dilution  the  green 
rays  likewise  appear.  Such  solutions,  therefore,  appear  green  when 
viewed  with  transmitted  light,  while  with  reflected  light  they  are 
red  and  fluorescent. 

When  a  fresh  leaf  is  similarly  examined,  a  spectrum  is  obtained 
which  is  essentially  the  same  as  that  just  described.  There  is  lack- 
ing, however,  the  band  that  corresponds  to  the  red  fluorescent  rays 
of  chlorophyl  solutions.  This  is  explained  by  the  assumption  that 
the  red  rays  are  absorbed  by  living  chlorophyl  and  transformed  into 
chemical  energy.  In  accordance  with  this  view,  we  find  that  when 
living  plants  are  successively  exposed  to  the  various  rays  constitut- 
ing sunlight,  decomposition  of  carbon  dioxide  with  liberation  of 
oxygen — which,  as  we  shall  presently  see,  takes  place  in  the  green 
portions  of  every  plant  whenever  it  is  exposed  to  sunlight — occurs 
with  special  intensity  when  the  plant  is  exposed  to  the  rays  corre- 
sponding to  the  bands  I,  II,  and  III.  In  this  manner,  then,  chloro- 
phyl-bearing  plants  derive  their  kinetic   energy  from  sunlight,  and 


THE  FOOD-STUFFS  OF  PLANTS.  23 

thus  become  enabled  to  elaborate  the  simple  food-stuffs  which  are 
at  their  disposal  into  the  complex  substances  which  constitute  their 
tissues. 

The  Food-stuffs  of  Plants.— The  most  essential  elements  which 
enter  into  the  composition  of  the  tissues  of  plants  are,  as  has  been 
pointed  out,  carbon,  hydrogen,  oxygen,  and  nitrogen.  These  sub- 
stances are  available  to  the  plant  as  carbon  dioxide,  water,  and  cer- 
tain nitrates.  The  origin  of  the  first  mentioned  is,  of  course,  obvious, 
while  that  of  the  last  is  at  first  sight  somewhat  obscure. 

The  nitrates  are  present  in  any  soil  which  contains  organic 
matter,  and  are  now  known  to  result  from  this  through  the  special 
activity  of  certain  bacteria.  Decomposing  animal  and  vegetable 
matter  is,  however,  not  the  only  source  of  the  nitrates,  for  it  can  be 
demonstrated  that  arable  soil,  apparently  devoid  of  vegetable  life  is 
capable,  unless  sterilized,  of  fixing  a  very  considerable  amount  of 
nitrogen,  which  must  of  necessity  be  derived  from  the  atmosphere. 
It  is  not  in  its  elementary  form,  however,  that  the  nitrogen  which  we 
thus  find  stored  reaches  the  soil,  but  in  all  probability  as  an  ammo- 
niacal  compound.  This  the  bacteria  then  transform  into  nitrates, 
which  the  plant  requires  for  its  existence.  I  do  not  wish  to  convey 
the  impression,  however,  that  all  plants  require  their  nitrogen  in  this 
form,  for  we  know  that  the  Saccharomyces  cerevisia?,  for  example, 
can  elaborate  its  nitrogen  not  only  from  ammoniacal  salts  directly 
but  is  even  incapable  of  utilizing  that  which  is  furnished  in  the 
form  of  nitrates.  Under  certain  conditions,  moreover,  probably  all 
plants  can,  for  a  while  at  least,  grow  in  the  presence  of  ammoniacal 
nitrogen  only. 

From  what  has  been  said,  it  is  clear  that  a  certain  symbiosis  exists 
between  the  bacteria  of  the  soil  and  plants,  in  virtue  of  which  the 
former  transform  the  ammoniacal  nitrogen  that  is  present  in  the 
soil  into  nitrates,  which  can  be  utilized  by  plants,  while  they  in  turn 
probably  aid  the  nutrition  of  the  bacteria  by  furnishing  them  with 
humus  and  the  ternary  matter  which  is  necessary  for  their  develop- 
ment. 

The  necessary  mineral  salts   the  plant  likewise  obtains  from  the 

.-oil. 

The  question  now  arises:  In  what  manner  do  plants  effect  the 
synthesis  of  those  complex  chemical  substances  which  go  to  form 
their  tissues  from  the  simple  bodies  which  serve  as  their  food-stuffs? 
The  kinetic  energy  which  is  necessary  to  effeel  these  changes  is,  as 
has  been  stated,  derived  from  the  sunlight  and  transformed  into 
potential  energy  by  the  chloropbyl.  We  should  thus  expect  to  find 
|"  those  part-  in  which  this  is  present  the  origin  of  those  final 
products  which  we  meet  with  in  the  tissues  of  the  plant.  These 
products  nmy  be  divided  into  three  groups,  and  in  the  following 
pages  ;m  attempt  will  he  made  to  describe  the  manner  in  which 
representatives  of  each  are  formed.  I  -hall  accordingly  consider 
the  origin  of  the  carbohydrates,  the  fats,  albumins, and  certain  non- 


24  •  INTRODUCTION. 

albuminous,  nitrogenous  bodies,  all  of  which  are  also  found  in  the 
animal  body,  and  which  represent  the  essential  food-stuifs  of  the 
animal  world.  In  doing  so,  I  am  aware  that  I  am  trespassing  to 
a  certain  extent  upon  what  will  follow  in  subsequent  chapters ;  but 
as  I  shall  deal  with  the  chemistry  of  animal  life  more  exclusively 
in  the  present  work,  it  has  been  deemed  best  to  consider  briefly  the 
principal  syntheses  which  are  effected  by  plants  before  proceeding  to 
a  more  detailed  study  of  the  subject  proper. 

Synthesis  of  the  Carbohydrates. — It  has  been  pointed  out 
that  during  exposure  of  chlorophyl-bearing  plants  to  sunlight 
the  carbon  dioxide  of  the  air  is  decomposed,  with  liberation  of 
oxygen.  The  volume  of  gas  thus  set  free  is  equivalent  to  the 
volume  of  carbon  dioxide  that  is  decomposed.  At  the  same  time  a 
reduction  of  water  takes  place,  as  is  apparent  from  the  observation 
that  a  larger  amount  of  hydrogen  is  found  in  the  plant  than  is 
necessary  to  form  water  with  all  of  the  oxygen  that  is  present  at  the 
same  time.  It  thus  follows  that  one-half  of  the  oxygen  per  volume 
must  be  derived  from  carbon  dioxide,  and  the  other  from  water, 
according  to  the  equation  : 

C02     +     H20     =     r     +     20, 
2  volumes.  2  volumes. 

in  which  r  represents  one  atom  of  carbon,  one  atom  of  oxygen,  and 
two  atoms  of  hydrogen,  which  have  been  retained  by  the  plant.  A 
combination  of  these  atoms  in  one  molecule,  however,  would  repre- 
sent one  molecule  of  formic  aldehyde,  CH20.  Should  this  be 
actually  formed  in  the  plant,  we  would  at  once  have  a  probable 
explanation  of  the  manner  in  which  the  carbohydrates  are  con- 
structed, as  these  are  polymeric  compounds  of  formic  aldehyde,  or 
their  anhydrides.  In  fact,  it  is  possible  artificially  to  effect  the 
synthesis  of  many  of  the  carbohydrates  which  are  found  in  the 
living  world  by  starting  with  this  simple  aldehyde.  Formic  alde- 
hyde, it  is  true,  has  not  as  yet  been  isolated  as  such  from  the  leaves 
of  plants,  as  its  existence  here  is  probably  only  momentary,  but  its 
oxidation-product,  formic  acid,  has  frequently  been  obtained.  It  is 
thus  extremely  probable  that  this  is  actually  the  starting-point  in  the 
elaboration  of  the  carbohydrates  by  the  plant.  As  to  the  exact 
manner  in  which  the  aldehyde  is  derived  from  carbon  dioxide  and 
water,  we  are  as  yet  uncertain ;  but  it  appears  from  the  interesting 
researches  of  Gautier  and  TimiriazefF  that  the  chlorophyl  first 
decomposes  water,  under  the  influence  of  sunlight,  and  forms  a 
hydride  of  chlorophyl,  which  is  colorless,  and  that  this  product 
subsequently  reduces  carbon  dioxide,  with  liberation  of  oxygen  and 
restitution  of  the  green  chlorophyl.  These  changes  may  be  rep- 
resented by  the  equations : 

(1)  x  +H20  =  xH2  +  0. 

(2)  *H2  +  C02  =z  +  H2CO  +  0. 


SYNTHESIS   OF  THE  CARBOHYDRATES.  25 

where  x  represents  the  chlorophyl.  As  a  matter  of  fact,  Gautier 
and  Timiriazeff  succeeded  in  obtaining  sneh  a  hydride — -protophyllin 
— which  turned  green  on  exposure  to  air,  or  in  an  atmosphere  of 
carbon  dioxide  under  the  influence  of  sunlight ;  while  in  the  dark, 
or  on  exposure  to  sunlight  in  an  atmosphere  of  hydrogen,  no  change 
occurred.  The  existence  of  this  body  in  etiolated  plants,  more- 
over, has  likewise  been  established. 

By  a  process  of  polymerization  and  subsequent  hydrolysis,  then, 
formic  aldehyde  gives  rise  to  the  large  number  of  carbohydrates 
which  are  found  in  the  vegetable  world.  Of  the  manner  in  which 
these  polymerizations  and  subsequent  changes  are  brought  about, 
however,  we  know  little  ;  but  there  is  reason  to  believe  that  they  are 
largely  effected  through  the  activity  of  special  ferments,  as  has  been 
indicated.  That  some  of  these  changes  take  place  in  the  chlorophyl- 
bearing  parts  of  the  plant  can  readily  be  demonstrated.  If  a  spiro- 
gvra.  for  example,  is  exposed  to  sunlight  after  having  been  kept  in 
darkness  for  some  time,  so  as  to  remove  any  sugar  that  may  have 
been  present  in  the  cell,  it  will  be  observed  that  after  a  very  few 
minutes  starch  granules  appear,  which  can  readily  be  detected  by  the 
addition  of  a  little  iodine  solution.  On  subsequent  removal  from 
the  light  the  starch  soon  disappears  from  the  green  parts  of  the 
plant,  and  is  carried  to  the  storage-cells  proper,  where  it  is  trans- 
formed into  dextrin,  glucose,  and  various  soluble  gums,  which  may 
be  further  transformed  into  celluloses,  certain  mucilages,  etc. 

Glucosides. — Closely  related  to  the  carbohydrates  proper,  the 
origin  of  which  has  just  been  considered,  is  a  group  of  substances 
which  likewise  occur  widely  distributed  in  the  vegetable  kingdom. 
These  are  the  so-called  glucosides.  They  are  so  termed  from  the 
fact  that  glucose  is  invariably  formed  during  their  hydrolytic 
decomposition,  which,  as  an  anhydride,  thus  constitutes  an  integral 
part  of  their  molecule.  This  observation  at  once  suggests  their 
origin  also  from  formic  aldehyde. 

Such  substances  are  salicin,  which  on  hydrolytic  decomposition 
yields  glucose  and  saligenin  ;  arbutin,  which  yields  glucose  and 
hydroquinon  ;  phloridzin,  which  gives  rise  to  glucose  and  phlorc- 
tin,  etc. 

(i)   c13h19ot  +  n./>    =  c,h8o2    +  cyi,2o6. 

Salicin.  Saligenin.  oinuose. 

(2)  f„irlfio7   +  h2o       c6ii6o,     i    c6n„<v.. 

Arhurtin.  Hydroquinon.       ulucose. 

(3)  C2]U2iOhl         II,o         C«HM06         C,H,A. 
Pnlondzin,  Pnloretin.  Glucose. 

Especially  interesting  is  a  group  of  glucosides  which  are  nitro- 
genous in  character,  and  thus  stand,  as  it  were,  midway  between  the 
carbohydrates  and  the  albumins.  A  study  of  their  decomposition- 
products  hence  permits  an  insight  into  the  manner  in  which  the 
albumin-  are  synthetically  produced,  and  show-  that  here  also  alde- 
hyde groups   play  an    important    part.      As  in    the   case  of  the  albu- 


26  INTR  OD  TJCTION. 

mins,  the  nitrogen  here  also  occurs  in  combination  with  carbon  and 
hydrogen  in  the  group  CH — JSTH,  which  in  turn  is  structurally  closely 
related  to  hydrocyanic  acid.  In  accordance  with  these  considera- 
tions, we  thus  find  that  amygdalin,  C^H^NOn,  is  decomposed  into 
glucose,  hydrocyanic  acid,  and  the  essence  of  bitter  almonds.  Solanin, 
C43H70NO16,  similarly  yields  glucose  and  solanidin,  C25H39]SrO. 

Mannides. — Like  the  carbohydrates  proper,  the  mannides  or  nian- 
nitides,  which  also  occur  widely  distributed  in  the  vegetable  world, 
are  likewise  derived  from  the  aldehyde  radicle  that  is  formed  by 
chlorophyl  under  the  influence  of  sunlight.  They  differ  from  the 
glucosides  in  yielding  mannite,  C6H1406,  instead  of  glucose,  on 
hydrolytic  decomposition.  The  origin  of  mannite  from  formic  alde- 
hyde may  be  represented  by  the  equation  : 

6CH20  +  2H  =  C6HM06. 

On  the  other  hand,  mannite  may  result  from  glucose  as  the  result 
of  the  specific  activity  of  certain  cells,  as  is  shown  by  the  equation  : 

13C6H1206  +  6H20  =  12C6HU06  +  6C02. 

Synthesis  of  the  Fats. — The  fats  which  are  found  in  plants 
are,  like  the  carbohydrates,  derived  from  carbon  dioxide  and  water, 
and  in  all  likelihood  are  formed  also  synthetically  through  the 
agency  of  the  chlorophyl.  The  mechanism,  however,  by  which 
these  syntheses  are  effected  is  not  so  clear.  It  is  probable  that  they 
result  from  the  union  of  carbon  dioxide  and  water,  as  shown  by  the 
equations : 


(1) 

3C02 

+  4H20 

=  C3H803 

Glycerin. 

+  70, 

(2) 

34C02 

+  34H?0 

=  C|8H3602 
Stearic  acid. 

+  16CH202  +  680. 
Formic  acid. 

(3)     C3H5(OH)3  +  3C18H35O.OH  =  C3H5(O.C,8H350)3  +  3H20. 
Glycerin.  Stearic  acid.  Stearin. 

This  supposition  is  strengthened  by  the  observation  that  during 
certain  phases  in  the  life  of  some  plants  an  actual  transformation 
of  carbohydrates  into  fats  takes  place.  In  the  fruits  and  leaves  of 
the  olive  tree,  for  example,  a  large  amount  of  mannite  gradually 
disappears  during  the  months  of  September  and  October,  and  is. 
replaced  by  oil.  This  transformation  could  be  explained  upon  the 
basis  of  the  equations  just  given,  or  by  the  assumption  that  the  oil 
results  from  mannite  through  a  loss  of  water  and  carbon  dioxide,  as 
suggested  by  the  equation  : 

11C6HU06  =  C51H9406  +  30H2O  +  15C02. 

In  any  event,  the  system  H20  -f  C02,  which  gives  rise  to  the 
formation  of  formic  aldehyde  and  glucose,  must  also  be  regarded 
as  the  fundamental  basis  in  the  synthesis  of  fats. 

Synthesis  of  the  Albumins. — Much  more  complicated  than  the 
synthesis  of  the  carbohydrates  and  fats  is  that  of  the  albumins, 


SYS THESIS  OF  THE  ALBUMINS.  27 

a  class  of  bodies  which  occur  widely  distributed  in  both  the  animal 
and  the  vegetable  world,  and  form  the  groundwork,  so  to  speak,  of 
all  living  matter.  Like  the  carbohydrates  and  fats,  they  also  con- 
sist of  carbon,  hydrogen,  and  oxygen,  but  in  addition  to  these  ele- 
ments nitrogen  and '  variable  amounts  of  sulphur  are  constantly 
present.  To  this  class  belong  such  bodies  as  serum-albumin,  egg- 
albumin,  casein,  fibrin,  etc.  They  are  exceedingly  complex  sub- 
stances,  and  have  a  very  high  molecular  weight.  For  vitellin,  for 
example,  Bnnge  obtained  the  formula  C292H481N9(,O^S2,  which  would 
correspond  to  a  molecular  weight  of  at  least  7557. 

The  exact  manner  in  which  the  albumins  originate  has  not  been 
determined,  and  the  many  attempts  which  have  been  made  to  effect 
the  synthesis  of  bodies  belonging  to  this  class  have  been  fruitless. 
We  are  in  possession  of  a  number  of  observations,  however,  which 
permit  of  an  insight,  at  least,  into  the  manner  in  which  plants,  are 
capable  of  elaborating  these  complex  substances  from  the  simple 
material  which  serves  as  their  food,  and  there  is  reason  to  suppose 
that  the  synthesis  of  the  albumins  also  takes  place,  to  a  certain 
extent  at  least,  in  the  chlorophyl-bearing  portions  of  plants. 

It  was  formerly  supposed  that  the  nitrogen  necessary  in  these 
synthetic  processes  was  furnished  plants  in  the  form  of  ammoniaeal 
salts.  Subsequent  investigations  have  shown,  however,  as  has  been 
indicated,  that  this  is  usually  not  the  case,  and  we  now  know  that 
through  the  activity  of  various  bacteria  in  the  soil  the  nitrogen 
required  by  plants  is  here  oxidized  to  nitrates.  These  are  absorbed 
and  carried  to  the  chlorophyl-bearing  portions  of  the  plant,  where, 
as  we  have  seen,  formic  aldehyde  and  glucose  are  constantly  being 
formed.  Here,  or  in  the  rootlets,  a  certain  proportion  of  the  nitrates 
is  apparently  transformed  into  nitric  acid,  which  is  then  promptly 
reduced  by  the  formic  aldehyde,  with  the  formation  of  a  certain 
amount  of  hydrocyanic  acid,  as  shown  in  the  equation : 

2HNOs  +  5CH2  O  =  2HCN  +  3C02  +  5H20. 

In  this  form,  then,  the  nitrogen  probably  enters  into  the  construc- 
tion of  the  albuminous  molecule.  This  supposition  is  strengthened 
l>v  the  observation  that  hydrocyanic  acid,  as  Buch,  or  in  the  form  of 
cyanides,  occurs  widely  distributed  in  the  vegetable  world,  and  is 
characterized  by  the  readiness  with  which  it  combines  with  a  large 
Dumber  of*  organic  substances  to  form  highly  complex  chemical  coin- 
pounds.  A  study  of  the  decomposition-products  of  the  various 
albumins  further  shows  that  their   molecule  can   be  reduced  to  urea, 

/MI  CO— NIL 

(  ( )     vnJ,  -'"id  oxamide,    |  ,  the  hydrogen  atoms  of  which 

\.mi,  CO— NH2 

have     either    entirely    or    partly    been    replaced    by    the    chains 

ro—cn,—  fii,  —  en—  Nil— rir,—  CH— Nil — CH2—  <  <><>n. 

On  studying  this  chai ore  closelv.il   will  be  observed  thai   the 


28  INTB  OD  UCTION. 

formic  aldehyde  radicle  CH2  is  here  repeatedly  encountered,  and 
that  these  radicles  are  united  by  the  group  CH — NH,  which  in 
turn  represents  hydrocyanic  acid  plus  one  atom  of  hydrogen. 
This,  as  we  have  just  seen,  results  from  the  action  of  formic  alde- 
hyde on  nitric  acid.  Through  a  union  of  such  aldehyde  groups 
with  hydrocyanic  acid  chains  could  then  result  of  the  composition  : 

H    H  H     H 

III  II  I 

=  C-NH-C-C-C-NH-C-C-NH-C-C  =  M, 

III  II  I 

OHOH  OHOH 

in  which  through  a  process  of  hydration  the  first  and  last  C — NH 
groups  can  be  transformed  into  CO  and  COOH,  according  to  the 
equations  : 

HON  +  H20  =  CO  +  NH3  and  HCN  +  2H20  =  H.COOH  +  NH3. 

In  the  presence  of  nascent  hydrogen,  moreover,  the  aldehyde  and 
hydrocyanic  acid  groups  would  be  transformed  into  the  groups 
H  H 

II 

C   and    C  — N — ,  thus  giving  rise  to  the  chains : 

I  I 

H  H 

—  CO  —  CH2  —  CH2  —  CH.NH  —  CH2  —  CH2  —  CH.NH  —  CH2  —  COOH. 

That  hydrogen  is  actually  available  in  the  leaves  for  this  purpose 
we  know,  as  during  the  formation  of  formic  acid  from  its  aldehyde 
hydrogen  is  constantly  being  liberated. 

The  formation  of  urea  and  oxamide,  finally,  the  radicles  of  which, 
as  we  shall  see,  are  possibly  present  as  such  in  the  albuminous  mole- 
cule, could  further  be  explained  upon  the  basis  just  outlined,  as  we 
know  that  both  can  be  formed  by  hydration  from  hydrocyanic  acid, 
as  is  shown  by  the  equation  : 

2HCN  +  2H20  =  CO  (NH2)2  +  CH2  O. 

According  to  Gautier,  then,  these  chains  are  further  united  to 
tyrosin,  so  that  the  structural  formula  of  albumins  could  be  repre- 
sented by  the  general  formula : 

/COOH 

C,Hq 


NOH 
H— C 

^c      c 


1/ 


h/  1       1  \h 

E  v    I  |E 

)c       c( 

Hx    \  /    XH 
H— C 

I 
OH 

in  which  R  represents  the  chains  in  question. 


SYNTHESIS  OF  THE  ALBUMINS.  29 

Should  this  theory  as  to  the  origin  of  the  albumins  in  plants  prove 
correct,  it  would  thus  be  clear  that  all  three  of  the  great  classes  of 
food-stuffs  which  animals  require  for  their  existence  are  formed 
synthetically  under  the  influence  of  sunlight,  and  through  the 
special  activity  of  chlorophyl.  Subsequent  changes,  of  course, 
take  place,  whereby  the  albumins,  like  the  carbohydrates  and  fats, 
arc  transformed  into  those  peculiar  modifications  of  the  original 
compounds  which  are  required  by  the  various  organs  of  the  plant. 
These  changes,  however,  are  in  a  manner  of  only  secondary  impor- 
tance, and  not  to  be  compared  in  complexity  to  the  elaborate  syn- 
thetic processes  which  have  previously  occurred.  They  are  brought 
about  through  the  specific  activity  of  the  various  cells  of  the 
organism,  and  in  part,  at  least,  through  the  agency  of  ferments. 

The  food-stuffs  which  are  thus  elaborated  by  the  plants  cannot  all 
be  utilized  by  the  animal  as  such,  however,  and  previous  to  their 
assimilation  they  are  further  modified.  The  albumins  are  thus  trans- 
formed into  albumoses  and  peptones,  starch  is  inverted  to  maltose, 
and  the  fats  are  decomposed  and  saponified.  These  processes  of 
what  may  be  termed  primary  assimilation,  or  digestion,  render  the 
food-stuffs  capable  of  passing  through  the  mucous  membrane  of  the 
gastro-intestinal  canal.  During  this  passage  the  albumoses  and 
peptones  are  retransformed  into  albumins  proper,  maltose  is  changed 
into  glucose,  and  the  fats  are  reconstructed  from  their  two  com- 
ponents. Subsequently  all  these  bodies  are  further  modified  accord- 
ing to  the  character  of  the  tissues  in  which  they  are  to  be  utilized. 
Ultimately,  however,  they  give  rise  to  the  formation  of  those  simple 
substances  which  plants  require  for  their  existence — that  is,  into 
carbon  dioxide,  water,  and  certain  nitrogenous  bodies  which  readily 
give  rise  to  the  formation  of  amnion  iacal  salts. 

The  passage  through  the  body  of  the  various  elements  which  go 
to  form  the  tissues  and  organs  of  both  plants  and  animals,  and  the 
various  chemical  and  physical  changes  which  are  here  involved,  con- 
stitute the  phenomena  of  metabolism  ;  and  we  may  thus  state  that 
physiological  chemistry  deals  primarily  with  the  various  metabolic 
processes  which  occur  in  the  living  world. 

lief'ore  proceeding  to  a  study  of  these  various  changes  in  the 
animal  body,  however,  it  will  be  well  to  review  the  chemical  proper- 
tic-  and  the  composition  of  the  various  foot  1-st nil's  which  enter  into 
tin-  construction  of  its  tissues.  We  shall  accordingly  consider  the 
chemistry  of  the  albumins,  the  carbohydrates,  and  fats,  and  then 
attempt  to  follow  the  course  of  these  bodies  through  the  living 
organism  so  far  as  this  is  possible  with  the  present  state  of  our 
knowledge. 


CHAPTEK    II. 

THE  ALBUMINS. 

The  albumins,  or  proteins,  are  the  most  important  food-stuffs 
■which  animals  require  for  their  existence.  Albumins  enter  into  the 
construction  of  all  the  tissues  and  organs  of  the  body,  and  form  the 
groundwork  of  every  living  cell.  The  phenomena  of  life,  indeed, 
are  dependent  upon  and  centre  in  their  presence. 

While  many  different  forms  of  albumin  exist,  they  all  present 
certain  general  chemical  and  physical  characteristics  which  serve  to 
distinguish  them  as  a  class,  and  show  that  a  close  genetic  relation- 
ship exists  between  them. 

Elementary  Composition. — All  albumins  contain  carbon,  hydro- 
gen, nitrogen,  oxygen,  and  sulphur  in  certain  definite  proportions, 
which  vary  but  little  in  the  different  members  of  the  group.  The 
variations  which  occur  are  shown  in  the  following  table  : 

Carbon 50.0-55.0  per  cent. 

Hydrogen 6.5-  7.3       "       " 

Nitrogen 15.0-17.6       "       " 

Oxygen 19.0-24.0  "       " 

Sulphur       0.3-  2.4  "       " 

Other  elements  are  not  found  in  the  albumins  proper,  but  may 
occur  in  certain  compound  albumins,  which  are  formed,  through  the 
union  of  an  albuminous  group  with  other  more  or  less  complex 
radicles.  The  coloring-matter  of  the  blood  thus  contains  iron  ;  the 
most  important  constituents  of  the  nuclei  of  cells  are  more  or  less 
rich  in  phosphorus ;  other  bodies  belonging  to  this  order  contain 
iodine,  etc. 

All  albumins,  moreover,  contain  variable  amounts  of  mineral 
salts,  which  are  closely  bound  to  the  albuminous  molecule.  The 
most  important  and  constant  of  these  are  the  chlorides  and  phos- 
phates of  the  alkalies  and  the  alkaline  earths. 

Crystallization. — In  the  eggs  of  certain  fish  and  amphibia 
so-called  yolk-platelets  may  be  observed,  which  apparently  possess 
a  crystalline  structure.  Chemical  examination,  however,  has  shown 
that  these  bodies  do  not  consist  of  pure  albumins,  but  also  contain 
a  large  percentage  of  lecithins  and  mineral  salts.  The  so-called 
aleuron  crystals,  which  have  been  found  in  the  seeds  of  certain 
plants,  are  thus  likewise  not  composed  of  a  pure  albuminous  sub- 
stance, and  the  same  probably  holds  good  of  the  little  eosinophilic 
crystalloids  which  may  be  seen  in  the  blood  of  birds.     Artificially 

30 


DIFFUSION.  31 

also  the  crystallization  of  albumins  is  apparently  possible  only  in  the 
presence  of  certain  mineral  salts.  Numerous  attempts  to  bring  this 
about  in  the  absence  of  salts  have  so  far  at  least  yielded  only  nega- 
tive results.  Of  vegetable  albumins,  the  phytovitellin  of  para-nuts, 
the  castor-oil  bean,  etc.,  yields  well-defined  crystals  when  the  sub- 
stance is  dissolved  in  solutions  of  neutral  salts  at  a  temperature  of 
about  40°  C,  and  is  subsequently  allowed  to  cool  or  evaporate. 
Egg-albumin,  the  serum-albumin  of  the  horse,  and  pure  casein  may 
similarly  be  made  to  crystallize.  The  material  thus  obtained  does 
not  represent  pure  albumins,  however,  but  is  apparently  a  compound 
with  the  salts  employed.  The  tendency  to  crystallization,  moreover, 
increases  with  repeated  exposure  to  the  various  salt  solutions  in 
which  crystallization  is  to  take  place. 

The  globulins  have  thus  far  not  been  obtained  in  crystalline  form 
by  artificial  means,  but  Paton  has  shown  that  after  their  passage 
through  the  kidneys  they  may  at  times  separate  out  in  crystalline 
form  spontaneously. 

In  the  dry  state  the  albumins  usually  occur  in  the  form  of  a  white 
powder,  or  as  yellowish,  brittle,  more  or  less  opaque  lamellse,  which 
are  both  odorless  and  tasteless. 

Solubility. — Some  of  the  albumins,  such  as  serum-albumin  and 
egg-albumin,  are  soluble  in  water.  Others  are  insoluble  in  water, 
but  dissolve  in  dilute  saline  solution,  wThile  still  others  are  insoluble 
in  both  water  and  dilute  saline  solution,  but  dissolve  in  dilute  alka- 
line or  acid  solutions. 

All  albumins  are  soluble  in  concentrated  acetic  acid  and  in 
strong  solutions  of  the  caustic  alkalies,  but  in  undergoing  solution 
they  are  more  or  less  modified  and  transformed  into  syntonins  or 
alkaline  albuminates,  as  the  case  may  be  (see  below).  In  cold 
absolute  alcohol  and  ether  albumins  are  insoluble,  but  in  dilute 
alcohol  some  of  them  dissolve  with  comparative  ease. 

Behavior  toward  Neutral  Salts  and  Alcohol. — All  albumins, 
with  the  exception  of  certain  deutero-albumoses  and  peptones,  may 
be  precipitated  from  their  neutral  or  feebly  acid  solutions  by  satura- 
tion with  ammonium  sulphate.  Other  neutral  salts  of  the  alkalies 
and  alkaline  earths,  such  as  sodium  chloride  and  magnesium  siil- 
phate,  behave  in  a  different  manner  toward  the  individual  representa- 
tives of  the  group, and  it  is  thus  possible  nut  only  to  separate  the 
albumins  from  a  large  number  of  other  substances,  but  also  from 
each  other.  In  being  thus  precipitated  their  structure  and  proper- 
tie-  are  not  altered  iii  the  Least. 

Strong  alcohol  act-  in  very  much  the  same  manner,  but  it  is  to  be 

noted  that  after  prolonged  exposure,  and  especially  in  the  presence 
of  -.1I1-,  the  albumins  pass  over  into  the  coagulated  state,  and  then 
remain  refractory  to  all  neutral  solvents. 

Diffusion. —  Like  the  colloids  of  the  inorganic  world,  so  also  are 
the  albumin-  practically  incapable  of  diffusing  through  animal  mem- 
branes.    This  peculiarity  Graham  explained  by  the  assumption  that 


32  THE  ALBUMINS. 

such  bodies  do  not  occur  in  a  state  of  actual  solution.  This,  how- 
ever, is  not  necessarily  the  case,  and  it  is  more  likely  that  their 
inability  to  pass  through  animal  membranes  is  to  be  explained  by 
the  great  size  of  their  molecule.  This  property  of  the  albumins  is 
very  important,  as  it  enables  us  to  separate  these  bodies  from  a  large 
number  of  other  substances  which  may  be  present  in  the  same  solu- 
tion, and  to  some  extent  also  from  each  other. 

Coagulation. — It  has  just  been  stated  that  according  to  Graham's 
view  the  albumins  do  not  occur  in  a  state  of  actual  solution.  While 
this  may  be  questionable,  we  know  nevertheless  that  solutions  of 
these  substances  are  quite  unstable  and  possess  a  marked  tendency 
to  revert  to  the  solid  state.  In  this  respect  also  they  behave  very 
much  like  the  inorganic  colloids.  Thus,  when  a  solution  of  sodium 
silicate  is  added  to  a  large  excess  of  dilute  hydrochloric  acid  the 
silicic  acid  which  is  thus  formed  is  apparently  held  in  solution.  If 
then  the  excess  of  hydrochloric  acid,  together  with  the  sodium 
chloride  which  was  formed  during  the  reaction,  are  removed  by 
dialysis,  an  apparently  clear  solution  of  silicic  acid  remains  in  the 
dialyzer.  This,  however,  is  at  once  transformed  into  a  thick,  gelat- 
inous mass  when  a  small  amount  of  carbonic  acid  is  passed 
through  the  solution.  Some  of  the  albumins,  such  as  the  globu- 
lins, behave  in  much  the  same  manner.  In  undergoing  such 
changes  the  albumins  may  retain  their  original  properties  and 
structure,  or  they  may  be  altered  in  such  a  manner  that  they  are  no 
longer  soluble  in  the  original  neutral  media.  Then  they  are  said  to 
be  coagulated. 

The  phenomenon  of  coagulation  is  common  to  all  true  albumins, 
and  upon  this  property  the  ability  of  certain  forms  to  occur  in  a 
more  or  less  solid  state  in  the  tissues  of  the  body  is  no  doubt 
dependent.  With  this  statement,  however,  I  do  not  wish  to  convey 
the  idea  that  the  albumins  which  go  to  form  the  groundwork  of  such 
structures  as  connective  tissue,  cartilage,  and  the  like,  occur  in  a 
state  of  actual  coagulation,  analogous  to  that  which  can  be  brought 
about  through  the  influence  of  heat.  The  phenomenon  simply  indi- 
cates the  direction  which  we  shall  have  to  follow  in  seeking  for  an 
explanation  of  the  occurrence  in  the  tissues  of  living  animals  of  cer- 
tain albumins,  in  the  solid  or  semisolid  state. 

In  certain  groups  of  albumins,  such  as  the  albumins  proper  and 
the  globulins,  coagulation  is  brought  about  in  the  most  characteristic 
manner  through  the  influence  of  heat,  providing  that  the  solution 
presents  a  neutral,  or,  better,  a  feebly  acid  reaction.  If  the  reaction 
is  alkaline,  coagulation  is  not  complete,  and  in  the  presence  of  a 
certain  amount  of  free  alkali  or  an  alkaline  carbonate  it  may  not 
occur.  .  An  excess  of  organic  acids  similarly  prevents  coagulation, 
and  care  is  therefore  necessary  to  insure  only  a  feebly  acid  reac- 
tion when  it  is  desired  to  free  a  solution  from  all  its  coagulable 
albumins 

As  the  temperature  at  which  coagulation  of  the  various  albumins 


BEHAVIOR    TOWARD   POLARIZED   LIGHT.  33 

takes  place  varies  in  the  different  representatives  of  the  group, 
it  is  possible  thus  to  separate  them  from  each  other,  and  to  identify 
the  individual  forms.  A  certain  care,  however,  is  necessary  in  this 
process,  as  the  temperature  of  coagulation  for  any  one  albumin 
varies  within  fairly  wide  limits  according  to  the  concentration  and 
reaction  of  the  solution,  and  the  kind  and  amount  of  salts  that  may 
at  the  same  time  be  present. 

Denaturization. — It  has  been  pointed  out  that  coagulation  alters 
the  character  of  all  albumins  in  a  profound  manner,  as  is  evidenced 
by  the  fact  that  they  are  then  no  longer  soluble  in  the  usual  media. 
Of  the  changes  which  take  place  in  the  albuminous  molecule  during 
this  process  we  know  nothing.  When  once  coagulated,  however,  it 
is  only  possible  to  effect  their  solution  by  chemical  processes,  which 
are  calculated  to  bring  about  definite  changes  in  structure.  Aside 
from  their  digestion  with  ferments,  the  coagulated  albumins  may  be 
dissolved  by  treating  with  dilute  solutions  of  the  alkalies  or  mineral 
acids,  or  with  concentrated  organic  acids  under  the  influence  of  heat. 
They  are  thus  transformed  into  alkaline  albuminates  and  acid  albu- 
mins, or  syntonins.  The  changes  which  the  albumins  thus  undergo, 
Neumeister  has  termed  the  denaturization  of  albumins.  As  a  con- 
sequence, the  products  which  are  thus  formed  differ  not  only  from 
the  native  albumins  in  their  general  chemical  composition,  but  also 
in  their  properties.  They  are  thus  insoluble  in  neutral  solutions, 
but  dissolve  with  ease  in  solutions  of  the  alkaline  hydrates,  of  sodium 
carbonate,  and  in  hydrochloric  acid.  From  their  acid  solutions  they 
are  precipitated  by  saturation  with  sodium  chloride  or  ammonium 
sulphate.  To  undergo  this  change,  it  is  not  necessary,  however, 
that  the  native  albumins  have  been  previously  coagulated,  as  solu- 
tions of  the  albumins,  which  have  been  boiled  after  the  addition 
of  alkalies,  or  large  amounts  of  organic  acids,  and  have  thus 
been  prevented  from  undergoing  coagulation,  behave  in  the  same 
manner. 

Behavior  toward  Polarized  Light. — All  albumins  are  lawo- 
rotatory — /'.  r.,  they  turn  the  plane  of  polarized  light  to  the  left. 
A-  the  degree  of  rotation  varies  with  the  different  members  of  the 
group  and  the  amount  of  albumin  present,  it  is  thus  possible,  not 
only  to  identify  the  individual  bodies,  but  also  to  determine  the 
amount  present.  The  specific  rotatory  power  of  some  of  the  more 
important  representatives  of  the  group,  for  the  yellow  line  1  >,  is  as 
follow-  : 

albumin («)  D  33°-38° 

3     Mm  albumin "  56° 

Lactalbumin "  :«;°-T7° 

im-globulin .  "  .V.)°-75° 

Fibrinogen "  43° 

Syntonin  (from  mvosin) "  72° 

hi  (dissolved  in  magnesium  sulphate  solution)    .  "  80° 

Alkaline  nil. urn i. i:iic "  62.2° 

Various  albumoseti "         70°-80° 


3-4  THE  ALBUMINS. 

Color-reactions. — The  color-reactions  to  be  described  are  not 
exclusively  characteristic  of  the  albumins,  and  in  examinations  in 
this  direction  it  is  always  necessary  to  employ  a  number  of  these 
tests  before  drawing  conclusions  as  to  the  presence  or  absence  of 
an  albuminous  substance. 

1.  The  Xanthoproteic  Reaction. — This  reaction  depends  upon  the 
formation  of  certain  nitro-derivatives  when  albumins  are  treated 
with  concentrated  nitric  acid.  The  test  is  conducted  as  follows  :  A 
few  c.c.  of  the  solution  in  question  are  treated  with  a  few  drops  of 
concentrated  nitric  acid,  when  in  the  presence  of  certain  albumins  a 
white  flaky  precipitate  develops,  which  turns  yellow  on  boiling. 
With  other  forms  the  solution  remains  clear,  but  also  turns  yellow 
on  boiling.  If  in  either  case  ammonia  is  then  added  in  excess,  a 
deep-orange  color  results  which  is  very  characteristic. 

The  reaction  is  supposedly  dependent  upon  the  existence  in  the 
albuminous  molecule  of  a  certain  aromatic  radicle  or  radicles  belong- 
ing to  the  phenol  or  phenyl  group.  It  is  therefore  also  obtained 
with  tyrosin,  phenol,  cresol,  phenyl-acetic  and  phenyl-propionic 
acid,  as  also  with  leucin. 

2.  Millon's  Reaction. — This  is  apparently  referable  to  the  pres- 
ence of  the  oxybenzoic  acid  radicle  in  the  albuminous  molecule, 
and  is  accordingly  obtained  with  all  those  albumins  which  on  tryptic 
digestion  yield  tyrosin. 

The  reagent  is  prepared  as  follows  :  A  few  grammes  of  mercuric 
nitrate  are  treated  with  an  amount  of  water  that  is  just  sufficient  for 
their  solution.  Any  basic  salt  that  may  have  been  formed  is  dis- 
solved with  fuming  nitric  acid,  when  a  solution  of  sodium  acetate 
is  added,  drop  by  drop,  until  the  mixture  reacts  with  a  dilute 
solution  of  phenol,  as  described  below. 

The  test  is  conducted  by  adding  a  few  drops  of  the  solution  to  be 
examined  to  a  few  c.c.  of  the  reagent,  when  in  the  presence  of  albu- 
mins a  white  precipitate  is  formed,  which  turns  a  brick  red  on  the 
application  of  heat.  If  undissolved  albumins  are  examined  in  this 
manner,  they  are  transformed  into  brownish-red  flakes. 

3.  The  Reaction  of  Adamkiewicz. — This  reaction  is  now  thought 
to  be  referable  to  the  simultaneous  presence  in  the  albuminous  mole- 
cule of  a  carbohydrate  radicle,  together  with  the  aromatic  groups 
which  give  Millon's  reaction. 

The  test  is  conducted  as  follows  :  A  particle  of  the  dry  albumi- 
nous substance  is  dissolved  in  a  small  amount  of  glacial  acetic  acid 
by  the  aid  of  heat,  and  then  treated  with  one-half  its  volume  of 
concentrated  sulphuric  acid.  Immediately,  or  on  boiling,  a  violet 
color  develops,  and  the  fluid  at  the  same  time  becomes  slightly  fluo- 
rescent. The  test,  however,  is  not  altogether  reliable,  and  with 
albumoses  and  peptones  gives  a  positive  reaction  only  when  these 
are  present  in  concentrated  form. 

4.  The  Biuret  Reaction. — It  is  thought  that  this  reaction  is  depen- 
dent upon  the  presence  of  a  urea-forming  radicle  in  the  albuminous 


COLOR-REACTIONS.  35 

molecule,  a>  a  very  similar  reaction  is  obtained  with  urea,  and  is  in 
that  ease  referable  to  the  formation  of  biuret.  It  is  possible,  how- 
ever, that  its  occurrence  may  be  due  to  a  cyanic  radicle,  which  is 
likewise  contained  in  biuret,  and,  as  we  have  seen  in  the  preceding 
chapter,  hydrocyanic  acid  is  in  all  probability  intimately  concerned 
in  the  synthesis  of  albuminous  substances.  Both  hydrocyanic  acid 
and  hydrocyanuric  acid,  moreover,  give  this  reaction,  and  it  is  to  be 
noted  that  the  former  yields  the  purplish  color  of  the  albumoses, 
while  the  latter  gives  rise  to  the  violet  color  of  the  native  albumins. 
The  test  is  conducted  as  follows  :  A  few  c.c.  of  the  solution  to  be 
examined  are  treated  with  an  excess  of  a  strong  solution  of  sodium 
hydrate,  and  then  drop  by  drop  with  a  2  per  cent,  solution  of  copper 
sulphate.  In  the  presence  of  albumins,  with  the  exception  of  phy- 
tovitellin,  a  pure  violet  color  is  obtained,  while  with  albumoses  and 
peptones  a  rose  color  develops.  If  larger  amounts  of  albumin  are 
present,  the  reaction  is  obtained  without  difficulty.  Should  traces 
only  be  present,  great  care  must  be  taken  not  to  add  too  much  of  the 
copper  solution,  as  otherwise  the  violet  color  is  obscured  by  the  blue 
of  the  copper  solution.  Where  larger  amounts  are  present,  it  is 
necessary  to  add  more  of  the  reagent.  An  excess  of  neutral  salts, 
which  is  often  present  when  this  test  is  employed,  does  not  interfere 
with  the  reaction.  If  ammonium  sulphate  is  present,  however,  it  is 
advisable  to  use  a  large  quantity  of  the  sodium  hydrate  solution,  in 
order  to  bring  out  the  color.  Should  magnesium  sulphate  be  con- 
tained in  the  solution,  a  precipitate  of  magnesium  hydroxide  is 
formed,  and  is  allowed  to  settle.  Sodium  chloride  does  not  interfere 
with  the  reaction. 

5.  Boiling  with  Hydrochloric  Acid. — The  reaction  apparently 
depends  upon  the  formation  of  furfurol,  which  yields  a  violet 
color  when  brought  in  contact  with  some  other  substance  which 
is  formed  from  the  albuminous  molecule  at  the  same  time.  What 
thia  other  substance  is,  however,  we  do  not  know.  The  albuminous 
material,  best  after  extraction  with  hot  alcohol  and  subsequently  with 
ether,  is  boiled  for  several  minutes  with  concentrated  hydrochloric 
acid,  to  which  a  drop  of  concentrated  sulphuric  acid  has  been  added. 
The  albumin    passes    into   solution    and    a   deep-violet  color  results. 

6.  The  Sulphur-test. — The  albuminous  solution  is  heated  with 
an  excess  of  sodium  hydrate  in  the  presence  of  a  small  amount  of 
a<'<tate  of  lead.  At  tir-t  the  solution  turns  brown,  and  later  a 
precipitate  of  black  sulphide  of  lead  results. 

7.  Molisch's  Test. — The  reaction  is  referable  to  the  presence  of  a 
carbohydrate  group  in  certain  alhumins,  which  gives  rise  to  the 
formation  of  furfurol  on  treating  with  concentrated  sulphuric  acid. 
'flic  tesl  i-  conducted  as  follows:    A  -mall  amount  of  the  material  is 

treated  with  a  few  drops  of  a  15  per  cent,  alcoholic  solution  of 
o-naphtol,  and  with  I  or  2  c.c.  of  concentrated  sulphuric  acid.  In 
the  presence  of  a  carbohydrate  group  the  liquid  assumes  a  beautiful 
violet  color. 


36  THE  ALBUMINS. 

Precipitation  of  the  Albumins. — It  has  been  pointed  out  that 
with  the  exception  of  the  peptones  practically  all  albumins  can  be 
precipitated  from  their  neutral  or  feebly  acid  solutions  by  certain 
neutral  salts.  During  this  process  they  apparently  undergo  no 
alteration  in  structure  or  in  their  properties,  and  remain  soluble 
in  the  usual  neutral  media.  There  is  a  large  number  of  other  sub- 
stances, however,  which  while  they  also  precipitate  the  albumins, 
either  cause  their  coagulation  or  combine  with  them  to  form  com- 
pounds which  are  insoluble  in  water.  Some  of  these  reagents  are 
extensively  used  in  the  chemical  laboratory  for  the  purpose  of  test- 
ing for  albumins  in  various  solutions.  The  most  important  ones  are 
here  briefly  considered  : 

1.  The  mineral  acids,  viz.,  nitric,  hydrochloric,  sulphuric,  and 
metaphosphoric  acid.  The  most  important  of  these  is  nitric  acid, 
for  the  reason  that  it  does  not  redissolve  the  precipitated  albumins 
in  the  presence  of  neutral  salts,  even  if  an  excess  has  been  added 
and  the  mixture  is  boiled.  The  test  is  conducted  by  allowing  a  cer- 
tain amount  of  the  acid  to  flow  beneath  the  solution  to  be  tested, 
when  in  the  presence  of  albumin  a  white  ring  of  coagulated  albumin 
appears  at  the  zone  of  contact  (Heller's  test). 

Orthophosphoric  acid  can  be  employed  only  in  very  concentrated 
form. 

2.  The  salts  of  the  heavy  metals.  The  salts  which  are  usually 
employed  are  copper  sulphate,  ferric  chloride,  neutral  and  basic 
acetate  of  lead,  platinum  chloride,  mercuric  chloride,  silver  nitrate, 
uranium  acetate,  and  others.  In  combining  with  these  the  albumins 
act  as  weak  organic  acids;  they  thus  set  free  the  corresponding 
acids  of  the  salts  and  combine  with  the  metallic  oxides. 

Especially  important  are  the  salts  of  iron  and  lead.  If  ferric 
chloride  is  added  to  an  albuminous  solution  containing  an  excess  of 
sodium  acetate  until  a  distinct  red  color  is  obtained,  the  albumins 
are  completely  precipitated  on  boiling.  The  same  result  is  reached 
on  boiling  albuminous  solutions  with  hydroxide  of  lead  in  the 
presence  of  lead  acetate. 

Other  reagents  which  may  be  employed  for  the  purpose  of  testing 
for  albumins  are  the  following,  but  in  combining  with  these  the 
albumins  act  the  part  of  a  base : 

3.  Tannic  acid,  or  picric  acid  after  acidifying  with  acetic  acid. 

4.  Mercuropotassic  iodide,  bismuthopotassic  iodide,  phosphotung- 
stic  acid,  and  phosphomolybdenic  acid,  all  cause  the  complete  pre- 
cipitation of  albumins  in  the  presence  of  a  mineral  acid.  These 
reagents  are  further  utilized  very  extensively  as  precipitants  of 
organic  bases  in  general,  and  notably  of  the  vegetable  and  animal 
alkaloids. 

5.  Hydriodic  acid  in  the  presence  of  mercuropotassic  iodide ; 
the  albuminous  solution  is  previously  acidified  with  hydrochloric 
acid. 

6.  Potassium   ferrocyanide,   as  well  as  the  ferricyanide,   in   the 


DECOMPOSITION   OF  THE  ALBUMINS.  37 

presence  of  acetic  acid.  This  test  is  quite  commonly  used  for  the 
purpose  of  demonstrating-  the  presence  of  albumin  in  the  urine. 

7.  Trichloracetic  acid  in  2  to  5  per  cent,  solution  is  now  extensively 
used  for  the  purpose  of  testing  for  certain  albumins,  notably  serum- 
albumin,  but  it  does  not  cause  complete  precipitation  of  all  forms  of 
albumin. 

Decomposition  of  the  Albumins. — With  the  view  of  gain- 
ing an  insight  into  the  structural  composition  of  the  albuminous 
molecule,  a  careful  study  of  the  decomposition-products  of  the 
various  albumins  has  long  occupied  the  attention  of  investigators. 
These  products  vary  somewhat  with  the  method  of  decomposition 
which  is  employed,  but  there  are  certain  ones  which  are  almost  con- 
stantly met  with,  and  which  hence  may  be  regarded  as  essential 
constituents.  Especially  important  among  these  are  certain  amido- 
acids,  such  as  tyrosin,  leucin,  asparaginic  acid,  glutaminic  acid,  and 
glycocoll.  These  are  formed  from  the  native  albumins,  no  matter 
whether  their  decomposition  has  been  effected  by  superheated  steam, 
by  boiling  with  acids  or  alkalies,  or  by  means  of  the  so-called  pro- 
teolytic ferments.  The  nitrogen  which  is  thus  split  off  is  spoken  of 
as  mono-amino-nitrogen.  At  the  same  time  another  portion  is 
liberated  in  the  form  of  ammonia,  the  so-called  amido-nitrogen.  As 
tyrosin  is  an  amido-acid  of  the  aromatic  series,  viz.,  para-oxyphenyl- 
amido-propionic  acid,  while  leucin,  a-isobutyl-acetic  acid,  glycocoll  or 
amido-acetic  acid,  as  also  asparaginic  acid  and  glutaminic  acid,  viz., 
amido-succinic  acid,  and  amido-glutaric  acid,  respectively,  belong  to 
the  fatty  series,  we  may  conclude  that  the  albuminous  molecule  con- 
tains aromatic  as  well  as  fatty  acid  radicles.  In  accordance  with 
this  view,  we  find  that  all  albumins  which  yield  tyrosin  on  tryptic 
digestion  also  give  Million's  reaction,  and  we  know,  furthermore, 
that  the  amide  of  asparaginic  acid,  asparagin,  as  also  glutamin,  the 
amide  of  glutaminic  acid,  occur  widely  distributed  in  the  vegetable 
world.  Glutaminic  acid  itself  is  obtained,  together  with  asparaginic 
acid,  when  albuminous  substances  are  boiled  with  dilute  mineral 
acid-;.  At  the  same  time,  two  basic  substances  may  be  obtained, 
which  Drechsel  has  termed  lysatin  and  lysatinin,  and  which  are 
apparently  homologous  with  two  other  bodies  which  occur  widely 
distributee!  in  the  animal  world,  namely,  kreatin  and  kreatinin. 
Lysatin  and  lysatinin,  moreover,  like  kreatin  and  kreatinin,  yield 
area  among  their  products  of  decomposition,  which  shows  that  this 
body,  which  represents  the  final  product  of  the  normal  metabolism 
iii  mammals,  can  result  directly  from  the  original  albuminous  mole- 
cule through  a  simple  process  of  hydrolysis,  and  may  possibly  exist 
in  it  a-  such. 

Other  decomposition-products  which  may  be  obtained  apparently 
from  all  tine  albumins  are  the   three  hexon  bases,  arginin,  lysin, 

and    hi-tidin.      These   substances    in    turn    are  derived    from    certain 

pro  tarn  ins,  and  Koseel  claims  thai  a  protamin  radicle  is  present 
in  all  albumins,  and  gives   rise  to  the  violet  biuret  reaction,     The 


38  THE  ALBUMINS. 

nitrogen  which  exists  in  the  albuminous  molecule  in  this  form  we 
speak  of  as  diamino-nitrogen. 

We  know,  further,  that  sulphur  exists  in  the  albuminous  mole- 
cule in  at  least  two  forms,  as  one  portion  can  be  readily  split  off  on 
heating  with  dilute  solutions  of  the  alkalies,  as  hydrogen  sulphide, 
while  the  other  and  larger  portion  can  be  obtained  only  when 
destruction  of  the  albuminous  molecule  is  carried  much  further. 

In  addition  to  the  bodies  which  have  been  mentioned  above,  still 
others  have  been  obtained  on  decomposition  of  the  albumins,  such 
as  carbonic  acid,  oxalic  acid,  acetic  acid,  phenol,  indol,  skatol, 
methylmercaptan,  etc.  Some  of  these,  no  doubt,  result  from  the 
further  destruction  of  the  substances  just  considered,  while  others 
originate  from  atomic  groups  which  are  as  yet  but  little  known. 

A  few  years  ago,  Cohn  announced  the  observation  that  during  the 
decomposition  of  various  albumins  with  concentrated  hydrochloric 
acid  a  certain  pyridin  derivative,  dihydroxy-pyridin,  may  be 
obtained.  This,  however,  proved  erroneous,  and  Cohn  himself 
later  found  that  the  substance  in  question  was  a  piperazin  derivative, 
dibutyl-diethylene  diamin,  which  is  isomeric  with  a  certain  leucini- 
mid  that  can  be  obtained  from  one  of  the  leucins. 

Besides  these  various  radicles,  a  carbohydrate  group  also  appears 
to  be  present  in  the  albuminous  molecule,  and  may  be  demonstrated 
by  means  of  Molisch's  test.  Its  presence,  as  we  shall  see,  is 
extremely  important,  and  explains  the  observation  that  under  cer- 
tain pathologic  conditions  sugar  can  appear  in  the  blood  at  a  time 
when  no  carbohydrates  are  ingested  in  the  food. 

Attempts  to  gain  an  insight  into  the  construction  of  the  albumi- 
nous molecule  from  a  study  of  its  oxidation-products,  have  on  the 
whole,  not  yielded  encouraging  results,  but  it  may  be  mentioned  that 
Maly  apparently  succeeded  in  bringing  about  oxidation  of  albumins 
without  causing  their  destruction.  He  thus  obtained  a  substance 
which  he  termed  oxyprotonic  acid,  or  oxyprotosulphonic  acid,  and 
which  has  the  character  of  a  polybasic  acid.  It  is  apparently  closely 
related  to  the  original  albumin  from  which  it  is  derived,  but  a  re- 
arrangement of  certain  atomic  groups  appears  to  have  taken  place,  as 
the  sulphur,  for  example,  is  held  in  firm  combination  in  its  entirety, 
and  no  tyrosin  can  longer  be  obtained  on  decomposing  the  substance 
with  superheated  baryta-water,  for  example.  On  further  oxidation 
oxyprotonic  acid  is  transformed  into  peroxyprotonic  acid,  which  con- 
tains 34  per  cent,  of  oxygen,  as  compared  with  22  per  cent,  in  the 
case  of  the  mother-substance. 

Synthesis  of  the  Albumins. — The  synthesis  of  a  native  albumin 
from  the  elements  has  thus  far  not  been  accomplished. 

Molecular  Size  of  the  Albumins. — As  it  is  questionable  whether 
any  albumin  has  thus  far  been  obtained  in  chemically  pure  form, 
it  follows  that  it  is  scarcely  possible  to  give  formula?  which  express 
the  true  composition  of  these  bodies.  Attempts  to  determine 
this  from  an  analysis  of  their  compounds  with    metals    have    not 


STRUCTURAL   COMPOSITION   OF  THE  ALBUMINS.  8"(J 

led  to  uniform  results.  In  the  ease  of  vitellin,  in  which  the  nearest 
approach  to  actual  conditions  has  probably  been  made,  Griibler  deter- 
mined the  molecular  weight  as  8848,  from  which  Bunge  deduced 
the  formula  Cil2H+slX()0OsiS.,. 

That  the  size  of  the  molecule  is  very  large  in  all  albumins  can- 
not be  doubted.  Sabanejetf,  who  recently  attempted  to  determine 
this  for  egg-albumin  by  means  of  Raoult's  method,  which  is  based 
upon  a  determination  of  the  lowering  of  the  freezing-point,  obtained 
the  figure  15,000.  Whether  or  not  this  method  can  be  successfully 
utilized  in  the  determination  of  the  molecular  weight  of  all  albumins 
remains  to  be  seen. 

Structural  Composition  of  the  Albumins. — Of  the  structural 
composition  of  the  albumins  little  is  known  that  is  definite.  We 
have  seen  in  the  preceding  chapter  that  in  plants  the  primary  syn- 
thesis of  the  albumins  probably  occurs  through  a  union  of  the 
radicles  of  formic  aldehyde  and  of  hydrocyanic  acid  to  form  chains 
of  the  composition 

I  I 

— CO— < '  1 1 ,.  CI  I ,— CI  I— XH— CH  —  CH— X  H— CH2— COOH, 

and  it  was  pointed  out  that  on  hydrolytic  decomposition  of  all  albu- 
min- certain  amido-acids,  belonging  to  the  fatty  acid  and  the  aro- 
matic scries,  are  constantly  obtained.  We  have  evidence,  moreover, 
that  a  protamin  radicle,  a  carbohydrate  group,  and  certain  sulphur 
groups  arc  present,  but  of  the  manner  in  which  these  various  groups 
arc  united  with  each  other,  and  of  their  distribution  in  the  albu- 
minous molecule,  we  know  practically  nothing. 

According  to  Schiitzenberger,  all  albumins  are  essentially  very 
complex  ureids,  or  oxamids,  in  which  the  urea  is  united  with  certain 
glucoproteins.  These  latter  on  hydrolytic  decomposition  take  up 
water,  and  form  amido-acids  of  the  leucin  and  leucein  scries,  re- 
spectively. They  may  be  represented  by  the  general  formulae 
< '  I !_,,  ,>*'<  )_,  and  CnE^„_lN02.  After  decomposition  the  nitrogen 
would  accordingly  be  found  as  amido-nitrogen,  while  in  the  albu- 
minoid molecule  itself  if  is  supposedly  present  as  imido-nitrogen. 

This  theory  is  based  essentially  upon  the  observation  that  during 
the  decomposition  of  the  albumins  with  superheated  baryta-water, 
carbon  dioxide,  oxalic  acid,  and  ammonia  are  formed  in  the  same 
relative  proportions  as  during  the  decomposition  of  urea  and 
oxamid. 

With  fibroin  Schiitzenberger  thus  obtained  the  following  complex 
result  : 

-     II     \  ,«>-.       24H20       5QHA       ll<<>        5C2H4Oa 
Fibroin.  Oxalic  acid.  lei  I  Ic  add. 

ami  the  mixture  of  amido-acids  yielded 

,  N'W,      C,HnNO,      7<  ,||.,\o,      7'    II, \<>,      2C4H„NOa       IC4H7N0 
Ty  rosin.  Glycocoll.  Alanin.  Amido  Amido-acid 

butyric  acid,    of  i  he  acrylic 
plea. 


40 


THE  ALBUMINS. 


The  theory  is  ingenious,  but  open  to  many  objections,  upon  which 
it  is  not  necessary,  however,  to  enter  at  this  place. 

Latham  regards  the  living  albumins  as  consisting  of  a  chain  of 
cyanic  alcohols  which  are  united  to  a  benzol  radicle.  Such  alcohols 
are  formed  through  union  of  an  aldehyde  with  hydrocyanic  acid, 
and  we  thus  see  that  his  idea  of  the  composition  of  the  albumins  is 
essentially  the  same  as  that  originally  suggested  by  Gautier.  The 
formation  of  the  various  decomposition-products  of  the  albumins 
Latham  explains  on  the  basis  of  the  extreme  instability  of  these 
compound  alcohols. 

Kossel,  on  the  other  hand,  divides  the  albumins  into  four  classes, 
and  assumes  that  in  each  a  protamin  radicle  is  the  essential  nucleus. 
Those  bodies  in  which  this  is  present  by  itself  he  assigns  to  the  first 
group,  and  it  is  accordingly  represented  by  the  protamins  them- 
selves. The  second  group  is  formed  by  albumins  in  which  the 
primary  protamin  nucleus  is  variously  combined  with  mono-amiclo- 
acids  of  the  aliphatic  series,  viz.,  with  leucin,  amiclo-valerianic  acid, 
asparaginic  acid,  glutaminic  acid,  and  glycocoll.  Most  of  these 
contain  in  addition  sulphur  in  more  or  less  intimate  combination, 
and  in  some  iodine  and  other  elements  also  may  be  found.  Other 
albumins  contain  an  aromatic  radicle  in  addition  to  the  pro- 
tamin group  and  the  acid  radicles  of  the  fatty  series,  and,  ac- 
cordingto  the  absence  or  presence  of  a  sulphur  group,  he  fur- 
ther divides  these  into  two  classes,  his  third  and  fourth  group, 
respectively.  Through  a  union  of  any  two  or  more  of  these 
groups  with  each  other,  or  with  new  prosthetic  groups,  still  more 
complicated  albumins  result,  such  as  the  histons  and  the  common 
proteids. 

Classification  of  the  Albumins. — The  various  albumins  may 
be  divided  into  four  classes,  viz.,  the  albumins  proper,  the  pro- 
teids, the  albuminoids,  and  what  may  be  termed  the  derived 
albumins.  They  are  further  subdivided,  as  is  shown  in  the  follow- 
ing table  : 


Albumins 


The  native  albumins    1    ~,  ,    , 

*    (jrlobul 


ins 


^  Vitellins 


f  Serum-albumin. 
J   Egg-albumin. 
J   Lactalbumin. 
^  Vegetable  albumin. 

Fibrinogen. 
Serum-globulin. 
Fibrinoglobulin. 
Vegetable  globulins. 
Myosin. 

f  Phytovitellin. 
\  Crystallins. 


i 


THE  NATIVE  ALBUMINS.  41 


f  Xucleins. 

The  proteids J   Nueleoproteids. 

j   (jlucoproteids. 
^  Haemoglobins. 

f  Keratins. 

The  albuminoids <   Albumoids. 

^  Amyloids. 


The  derived  albumins 


r  Albumoses. 

Peptones. 

Albuminates, 
j   Coagulated  albumins. 
^  Fibrin. 


THE  NATIVE  ALBUMINS. 

These  have  been  described  in  a  general  way  in  the  foregoing 
pages.     They  are  subdivided  as  above  indicated. 

The  Albumins. — The  albumins,  in  the  narrower  sense  of  the 
term,  comprise  serum-albumin,  egg-albumin,  lactalbumin,  and  the 
so-called  vegetable  albumin.  They  are  all  soluble  in  water,  but  may 
be  precipitated  from  their  neutral  aqueous  solutions  by  salting  with 
ammonium  sulphate.  Sodium  chloride  and  magnesium  sulphate 
cause  their  precipitation  only  if  the  solution  has  been  acidified  with 
acetic  acid.  The  addition  of  small  amounts  of  acids  or  alkalies  to 
their  aqueous  solutions  is  without  effect.  Larger  amounts  of  mineral 
arid-,  as  also  the  salts  of  the  heavy  metals,  cause  their  precipitation. 
Coagulation  occurs  on  boiling,  and  in  the  presence  of  a  certain 
amount  of  neutral  salts,  while  this  does  not  occur  if  the  solution 
contains  only  a  trace  of  salts. 

The  albumins  of  this  order  are  very  rich  in  sulphur,  containing 
from  1.6  to  2.2  per  cent.  The  nitrogen  is  held  in  part  as  so-called 
amido-nitrogen,  partly  as  diamino-nitrogen,  and  partly  as  tnono- 
amino-nitrogen. 

The  Globulins. — These  comprise  serum-globulin,  fibrinogen, 
fibrinoglobulin,  myosin,  and  various  vegetable  globulins.  They  are 
all  soluble  in  dilute  solutions  of*  the  neutral  salts,  and  may  be  pre- 
cipitated from  these  solutions  by  saturation  with  magnesium  sul- 
phate or  by  50  per  cent,  saturation  with  ammonium  sulphate. 
Sodium  chloride  precipitates  them  only  in  part.  Some  of  them  are 
insoluble  in  water,  while  others  are  soluble  without  difficulty.  II"  a 
dilute  saline  solution  of  the  common  serum-globulin  of*  the  blood- 
plasma,  for  example,  i-  subjected  to  dialysis,  a  certain  portion  of  the 
globulin  i-  precipitated.  Another  portion,  however,  remains  in 
solution,  and  may  lie  demonstrated  By  saturating  with  magnesium 
sulphate  or  by  saturation  with  60  per  cent,  ammonium  sulphate.     It 


42  THE  ALBUMINS. 

is  to  be  noted,  moreover,  that  the  portion  which  remains  in  solution 
represents  from-three-fifths  to  four-fifths  of  the  entire  amount  that 
was  originally  present,  but  it  appears  that,  barring  their  different 
solubility  in  water,  both  portions  are  identical. 

Some  of  the  globulins  may  also,  in  part  at  least,  be  precipitated 
from  their  neutral  solutions  by  copious  dilution  with  water,  by  pass- 
ing a  current  of  carbon  dioxide  through  the  solution,  or  by  acidifying 
with  acetic  acid  or  some  other  organic  acid.  If  an  excess  of  the 
acid,  however,  is  added,  they  again  dissolve.  All  globulins  are 
coagulated  by  heat,  and  it  is  to  be  noted  that  the  greater  number 
also  pass  into  the  coagulated  state  when  kept  long  under  water. 

Like  the  true  albumins,  the  globulins  contain  nitrogen  in  at  least 
three  forms,  viz.,  as  amido-nitrogen,  as  diamino-nitrogen,  and  as 
mono-amino-nitrogen.  They  contain  less  sulphur  than  the  albu- 
mins, but  not  less  than  1  per  cent. 

The  Vitellins. — The  vitellins  are  apparently  closely  related  to 
the  globulins  and  the  albumoses.  Some  of  them,  such  as  the 
aleurons  of  seeds  and  the  so-called  yolk-platelets,  which  are  found 
in  the  eggs  of  certain  fish  and  amphibia,  occur  in  crystalline 
form,  and  still  others  may  also  be  made  to  crystallize  artificially. 
As  has  been  mentioned,  these  crystalline  bodies  do  not  represent 
the  pure  albumins,  however,  but  are  probably  compounds  of  albu- 
mins with  various  salts  and  lecithins. 

Both  animal  and  vegetable  vitellins  are  soluble  in  dilute  saline 
and  alkaline  solutions ;  they  are  precipitated  from  these  by  acidify- 
ing with  dilute  acetic  acid,  by  passing  a  current  of  carbon  dioxide 
through  the  solutions,  and  by  salting  with  magnesium  sulphate  or 
sodium  sulphate  to  saturation.  Unlike  the  globulins,  they  cannot 
be  precipitated  from  their  solutions  by  saturation  with  sodium 
chloride. 

The  vitellins  proper  do  not  contain  phosphorus,  as  is  frequently 
stated,  but  in  the  eggs  of  birds  and  fish  they  are  commonly  found 
in  combination  with  lecithins  and  nucleins,  both  of  which  are  rich 
in  phosphorus.  Like  the  common  albumins  and  globulins,  they 
contain  also  sulphur,  but  in  variable  amounts. 

THE  PROTEIDS. 

The  proteids  differ  from  the  albumins  in  being  more  complex 
bodies,  and  consist  essentially  of  an  albuminous  radicle,  which  is 
variously  combined  with  a  non-albuminous  group.  This  may  be  of 
the  nature  of  a  phosphoric  acid  radicle,  or  a  carbohydrate  group,  or 
a  pigment.  In  this  manner  the  nucleins,  the  glucoproteids,  and  the 
haemoglobins  result.  The  nucleo-albumins,  further,  which  also 
belong  to  this  group,  are  formed  through  the  union  of  an  albu- 
minous radicle  with  a  nuclein. 

The  Nucleins. — The  nucleins  differ  from  the  true  albumins 
in   containing,  in   addition   to   carbon,  hydrogen,  nitrogen,  oxygen, 


THE  PROTEIDS.  43 

and  sulphur,  a  variable  amount  of  phosphorus,  and  in  some  instances 
also  iron 

Their  quantitative  composition,  moreover,  is  different,  as  is  ap- 
parent from  the  following  table  : 

Yolk-nuclein.  Veast-nuclein. 

Carbon 42.11  40.81 

Hvdrogen 6.08  5.38 

Nitrogen 14.73  15.98 

Oxygen 31.05  31.26 

Sulphur .    .    .    .      0,-,:,  0.38 

Phosphorus 5.19  6.19 

Iron 0.29  .    . 

The  nucleins  occur  widely  distributed  both  in  the  animal  and  the 
vegetable  world,  and  are  of  special  importance  as  food-stuffs,  in  so 
far  as  the  iron  which  some  contain  is  only  accessible  to  animals 
in  this  form.  They  are  essentially  albumins  which  are  closely  com- 
bined with  a  phosphoric  acid  radicle.  In  certain  forms,  however, 
this  group  is  not  only  united  to  an  albuminous  radicle,  but  also  with 
certain  basic  substances,  such  as  adenin,  hypoxanthin,  guanin,  and 
xanthin.  These  bodies  belong  to  the  class  of  the  so-called  xanthin, 
alloxuric,  or  purin  bases,  and  in  combination  with  a  phosphoric  acid 
radicle  constitute  the  so-called  nucleinic  acids.  Individually  these 
various  bodies  will  be  considered  in  another  section  of  this  work, 
but  it  may  here  be  mentioned  that  the  nucleinic  acids  and  the 
nucleinic  bases  not  only  occur  in  the  animal  body  in  combination 
with  albumins,  but  also  as  such. 

According  to  the  combination  of  the  albuminous  group  with 
phosphoric  acid  only,  or  through  this  with  the  nucleinic  bases,  the 
nucleins  are  now  divided  into  two  groups,  viz.,  the  so-called  para- 
nucleiri8,  or  pseudonueleins,  and  the  nuclear  nucleins  proper. 

All  nucleins  possess  the  character  of  strong  acids.  They  are 
soluble  in  solutions  of  the  hydrates  of  the  alkalies,  less  readily  so 
in  dilute  solutions  of  the  alkaline  carbonates  and  in  concentrated 
hydrochloric  acid.  In  water  and  alcohol  they  are  for  the  most  part 
insoluble.  They  are  coagulated  by  heat,  as  also  by  alcohol,  and  are 
then  insoluble  in  solutions  of  the  alkaline  hydrates.  In  dilute  acids 
and  in  artificial  gastric  juice  they  are  practically  insoluble,  and  it  is 
thus  possible  to  separate  them  from  any  albumins  that  may  be 
present  at  the  same  time. 

Like  the  albumins  proper,  they  give  the  various  color-reactions 
which  are  characteristic  of  the  albumins  as  a  class. 

The  Nucleo-albumins. — The  nucleo-albumins  are  compounds  of 
the  aucleine  and  paranucleins  with  a  special  albuminous  radicle. 
Like  the  nucleins,  they  hence  contain  phosphorus  but  their  quan- 
titative composition  varies  but  little  from  that  of  the  albumins 
proper.  This  is  no  doubt  owing  to  the  fad  thai  the  nucleinic  or 
paranucleinic  radicle,  which  enters  into  their  construction,  repre- 
sents only  a  -mall  portion  of  the  entire  molecule.     Like  the  nucleins, 

they  occur  widely  distributed   in   the  animal  and   vegetable  world, 


44  THE  ALBUMINS. 

and  are  of  special  importance  as  food-stuffs.  Some  of  them  also 
contain  iron.  They  possess  markedly  acid  properties,  and  can 
hence  combine  with  bases  to  form  salt-like  products.  Most  of  the 
nucleo-albumins  are  insoluble  in  distilled  water,  in  neutral  salt 
solutions,  and  in  weak  acids,  while  they  dissolve  with  ease  in  the 
presence  of  a  small  amount  of  an  alkaline  hydrate  or  lime-water. 

The  most  important  member  of  this  group,  casein,  occurs  in  solu- 
tion in  the  milk  as  a  calcium  compound.  In  combination  with  the 
alkalies  or  the  alkaline  earths,  the  nucleo-albumins  dissolve  in  water 
upon  the  application  of  heat,  and  it  is  to  be  noted  that  such  solu- 
tions do  not  coagulate  on  boiling.  Coagulation  occurs,  however,  as 
in  the  case  of  the  albumins  proper,  as  soon  as  the  basic  component 
is  removed  by  means  of  an  acid. 

Other  nucleo-albumins,  such  as  those  which  can  be  obtained  from 
the  yolk  of  birds'  eggs,  and  leucocytes,  are  soluble  in  dilute  acids  and 
in  a  10  per  cent,  saline  solution,  but  are  also  insoluble  in  water. 
From  their  solutions  they  are  partly  coagulated  by  heat.  Pepsin 
in  the  presence  of  0.2  per  cent,  of  hydrochloric  acid  decomposes 
the  nucleo-albumins  with  the  liberation  of  the  nucleins. 

The  Glucoproteids. — In  the  glucoproteids  an  albuminous  radicle 
is  combined  with  a  carbohydrate  group,  or  a  carbohydrate  deriva- 
tive which  may  or  may  not  be  nitrogenous.  They  all  contain  carbon, 
hydrogen,  nitrogen,  oxygen,  and  sulphur.  In  addition,  phosphorus 
has  been  found  in  certain  representatives  of  this  group,  and  these 
have  accordingly  been  termed  phosphoglucoproteids.  The  gluco- 
proteids proper  comprise  the  mucins,  the  mucoids  or  mucinoids, 
and  the  hyalogens,  all  of  which  are  peculiar  to  the  animal  world. 
Of  the  phosphoglucoproteids,  on  the  other  hand,  only  two  repre- 
sentatives are  known  at  the  present  time,  viz.,  the  ichthulin  of  carp 
eggs,  and  the  helicoproteid  which  may  be  obtained  from  the  albu- 
minous gland  of  Helix  pomata. 

The  carbohydrate  radicle,  which  may  be  separated  from  the  albu- 
minous group  on  boiling  with  dilute  mineral  acids,  is  apparently  not 
the  same  in  all  glucoproteids,  and  in  most  cases  its  true  chemical 
nature  has  not  as  yet  been  ascertained.  From  certain  mucins, 
Landwehr  claims  to  have  obtained  the  so-called  animal  gum, 
which  is  a  dextrin-like  carbohydrate,  when  the  substance  was  ex- 
posed to  the  action  of  superheated  steam.  On  carrying  the  decom- 
position further,  as  on  boiling  with  strong  mineral  acids,  lsevulinic 
acid  was  found,  besides  leucin,  tyrosin,  and  other  bodies  of  this 
order. 

From  the  helicoproteid  Hammarsten  succeeded  in  splitting  off  a 
gum-like  dextrorotatory  substance,  which  he  regards  as  animal 
sinistrin. 

Beyond  these  few  data,  however,  practically  nothing  is  known  of 
the  character  of  the  reducing  substance. 

While  all  glucoproteids  show  a  structural  composition  which 
warrants  their  classification  as  a  separate    group    of   proteids,  the 


THE  PROTEIDS.  45 

subgroups  differ  from  each  other  in  many  respects,  and  the  differ- 
ent representatives  of  each  group,  moreover,  possess  certain  features 
which  serve  to  distinguish  them  from  each  other. 

The  mucins  proper,  which  include  the  mucin  that  is  furnished  by 
the  large  mucinous  glands,  the  mucin  that  is  found  in  tendons  and 
the  umbilical  cord,  that  which  is  secreted  by  snails,  and  that  found 
in  the  capsule  of  frogs'  eggs,  are  insoluble  in  water.  They  possess 
acid  properties,  and  dissolve  in  water  after  neutralization  with  an 
alkali.  Such  solutions  do  not  coagulate  on  heating,  but  are  precip- 
itated on  acidifying  with  acetic  acid.  This  precipitate  is  insoluble 
in  an  excess  of  the  acid.  In  the  presence  of  from  5  to  10  per  cent, 
of  sodium  chloride,  however,  they  are  not  precipitated  in  this 
manner.  From  such  acid  solutions  they  are  not  thrown  down  by 
potassium  ferrocyanide,  while  tannic  acid  causes  the  mucin  to  sepa- 
rate out.  Neutral  solutions  of  the  mucins  are  precipitated  by 
alcohol  in  the  presence  of  neutral  salts.  Similar  results  are  ob- 
tained with  some  of  the  salts  of  the  heavy  metals.  When  heated 
on  a  water-bath  with  dilute  hydrochloric  acid  (2  per  cent.)  the 
mucins  are  decomposed,  with  liberation  of  the  carbohydrate  group, 
which  can  be  demonstrated  with  Fehling's  test  (see  Urine).  Ac- 
cording to  more  modern  investigations,  however,  it  appears  that  the 
radicle  which  is  thus  split  off  is  not  a  true  carbohydrate.  Miiller 
and  Seemann  were  thus  able  to  isolate  a  crystalline  substance  from 
mucins  which  was  apparently  identical  with  glucosamin,  and  Leathes 
obtained  a  body  which  he  regards  as  a  reduced  chondrosin.  Levene 
concludes  from  his  recent  investigations  that  the  mucins  contain  the 
complex  of  chondroitin-sulphuric  acid.  This  would  account  in  a 
satisfactory  manner  for  the  acid  properties  of  the  mucins,  and  the 
fact  that  they  yield  a  reducing-substance  on  decomposition.  This, 
however,  is  not  a  carbohydrate  proper,  but  glucosamin. 

All  mucinous  solutions  are  more  or  less  viscid,  and  are  therefore 
extremely  difficult  to  filter.  In  the  dry  state  they  area  white  or 
yellowish-gray  powder. 

The  mucoids,  or  mminoids,  are  found  in  the  cornea  and  the 
vitreous  humor  of  the  eye,  in  the  white  portion  of  birds'  eggs,  in 
cartilage,  and  arc  very  abundant  in  certain  ovarian  cysts,  where  two 
distinct  varieties  have  been  encountered,  viz.,  the  so-called  metalbu- 
min  or  paralbumin,  or  pseudomucin,  and  colloid.  They  will  be  con- 
sidered in  detail    later. 

The  hyalogem  comprise  ;i  class  of  substances  which,  according  to 
Krukenberg,  are  essentially  characterized  by  the  fact  that  on  treat- 
ment with  alkalies  they  are  decomposed  into  an  albuminous  sub- 
stance and  into  nitrogenous  carbohydrate-like  bodies,  the  so-called 
hyaliri8,  which  in  turn  are  gaid  to  yield  a  carbohydrate  proper  on 
further  decomposition. 

Among  the  hyalogens  may  be  mentioned  the  neossin  which  is 
found  in  edible  Chinese  swallow  nests;  membranin,  obtained  from 
I'      emet'fi    membrane   and    the   capsule   of  the   crystalline  lens; 


46  THE  ALBUMINS. 

spirograph  in,  from  the  spirographic  membrane ;  the  holothurian 
mucin  •  the  chondrosin  of  certain  mushrooms ;  and  others.  The 
hyalin  which  is  found  in  echinococcus  cysts,  and  the  onuphin  of 
the  tubes  of  Onuphis  tubicola,  which  have  both  been  regarded  as 
hyalogens,  are  apparently  not  proteids.  Hyalins  can,  however,  also 
occur  as  such,  or  as  closely  related  bodies  which  are  not  combined 
with  an  albuminous  group.  They  are  principally  found  in  the 
extra-skeletal  or  intra-skeletal  parts  of  various  animals.  Among 
these  may  be  mentioned  the  so-called  chondroitin,  which  occurs  in 
the  matrix  of  the  cartilage  of  the  higher  animals,  as  chondroitin- 
sulphuric  acid ;  and  chitin,  which  forms  the  greater  portion  of  the 
carapace  of  the  arthropods  and  the  inner  skeletal  structures  of  cer- 
tain cephalopods  and  brachiopods.  From  both  these  substances 
glucosamin  can  be  obtained  on  hydrolytic  decomposition. 

The  hyalogens  are  for  the  most  part  insoluble  in  water,  and,  as  we 
have  seen,  are  decomposed  by  treating  with  dilute  alkaline  solutions. 
They  give  the  general  color-reactions  of  the  albumins,  and,  like  the 
mucins  and  mucoids,  consist  of  carbon,  hydrogen,  nitrogen,  oxygen, 
and  sulphur.     Their  albuminous  radicles,  however,  are  unknown. 

Of  the  jyhosphoglucoproteids  little  is  known.  They  are  appar- 
ently related  to  the  nucleo-albumins,  and  yield  paranuclein  on  diges- 
tion with  artificial  gastric  juice. 

The  haemoglobins  are  essentially  compounds  which  contain  an 
albuminous  radicle  that  is  variously  combined  with  an  organic 
pigment.  They  will  be  considered  in  detail  in  the  chapter  on  the 
Blood. 

THE   ALBUMINOIDS. 

The  albuminoids,  as  they  are  commonly  termed,  are  closely  related 
to  the  albumins,  but  differ  from  these  in  many  important  par- 
ticulars. For  the  most  part,  they  contain  less  carbon  and  more 
oxygen  than  the  albumins  proper,  and  can  hence  be  regarded  as 
early  products  of  decomposition  and  oxidation.  They  are  not 
found  in  the  vegetable  world,  and  must  therefore  be  produced  in 
the  animal  body  through  a  certain  rearrangement  of  atoms  from 
the  vegetable  albumins."  During  the  reconstruction  of  the  molecule 
in  the  animal  body,  however,  a  certain  amount  of  carbon  is  mani- 
festly lost.  We  find,  as  a  matter  of  fact,  that  in  certain  representa- 
tives of  this  group  the  aromatic  radicle  is  lacking,  and  among  the 
decomposition-products  of  such  substances  we  accordingly  find 
neither  indol  nor  tyrosin.  Their  nutritive  value  is  therefore  also 
less  than  that  of  the  albumins,  and  Yoit  actually  demonstrated  that 
gelatin,  for  example,  is  in  itself  insufficient  to  maintain  life.  Certain 
members  of  this  group,  moreover,  cannot  be  regarded  as  food-stuffs 
at  all,  owing  to  the  extreme  resistance  which  they  offer  to  most 
solvents,  including  the  digestive  fluids. 

As  a  class  the  albuminoids  occur  widely  distributed  in  the  animal 


THE  ALBUMINOIDS.  47 

world,  and  form  the  greater  portion  of  the  internal  as  well  as  the 
external  skeleton.  The  most  important  members  of  the  group  are 
the  keratins,  the  principal  constituents  of  the  epidermal  structures  of 
the  animal  body;  elastin,  which  is  found  in  the  connective  tissue  of 
the  higher  animals  ;  collagen,  which  is  present  also  in  connective 
tissue  and  in  the  organic  portions  of  the  bones;  gelatin,  or  glutin, 
which  is  soluble  collagen,  viz.,  collagen  plus  water ;  the  skeletins, 
spongin,  conchiolin,  kornein,  fibroin,  sericin,  and  elastoidin,  which 
have  been  mentioned  as  occurring  among  the  invertebrate  animals  ; 
and,  finally,  the  so-called  amyloid  substance,  which  is  encountered 
under  various  pathologic  conditions.  Of  these  various  substances, 
the  keratins  and  elastin  are  more  closely  related  to  the  albumins 
proper  than  the  remainder.  They  both  give  rise  to  the  same 
decomposition-products  as  the  albumins,  though  elastin  yields  but 
very  little  tyrosin  and  no  glutaminic  or  asparaginic  acid.  They  give 
the  various  color-reactions  of  the  albumins,  but  it  appears  that 
elastin  only  contains  in  its  molecule  that  form  of  sulphur  which  is 
easily  split  off  on  boiling  with  dilute  alkalies.  Keratin,  on  the 
other  hand,  contains  much  sulphur — 3  to  5  per  cent. ;  and  it  is  inter- 
esting to  note  that  during  its  decomposition  a  fairly  large  proportion 
may  be  obtained  in  the  form  of  cystin.  Both  the  keratins  and 
elastin  can  be  brought  into  solution  only  by  means  of  superheated 
-team  or  by  boiling  with  strong  alkalies,  but  the  substance  is  at  the 
same  time  decomposed.  Concentrated  mineral  acids  also  dissolve 
elastin  with  varying  ease,  and  with  or  without  the  application  of 
heat,  according  to  the  origin  of  the  material. 

Collagen  and  its  hydrate  glutin,  or  gelatin,  on  the  other  hand,  are 
structurally  further  removed  from  the  true  albumins.  They  appar- 
ently contain  no  aromatic  radicle,  and  hence  on  decomposition  yield 
neither  tyrosin  nor  indol.  Leucin,  glycocoll,  glutaminic  acid,  and 
asparaginic  acid  are,  however,  always  obtained.  Pure  solutions  of 
gelatin  give  the  biuret  reaction  and  the  xanthoproteic  reaction,  while 
the  reactions  of  Millon  and  Adamkiewicz  are  negative.  The  sulphur 
is  apparently  present  only  as  closely  combined  sulphur,  as  hydrogen 
Bulphide  doe-  not  develop  on  boiling  with  dilute  alkalies. 

Solution-  of  collagen  gelatinize  on  cooling  and  redissolve  on  the 
application  of  heat.  The  behavior  of  the  substance  is  in  this 
respect  exactly  the  contrary  of  what  we  see  in  the  albumins  proper. 
The  mineral  acids,  potassium  ferrocyanide  in  the  presence  of  acetic 

acid,  and  -i  mineral  salts  do  not  precipitate  the  gelatin  from  its 

solutions. 

Solutions  of  cartilaginous  glutin,  which  was  formerly  termed 
c/io, uliin,  possess  characteristics  which  are  different  from  those  of 
glutin  thai  is  obtained  from  connective  tissue  or  decalcified  bones. 
These  differences,  however,are  not  owing  to  the  glutins  as  such,  but 
to  the   presence  of  certain  soluble  compounds  of  chondroitin-sul- 

(ilinric    acid,    the    diondroit in    radicle    01  which,    a-    we    have    seen, 

belongs  to  the  so-called  hyalins. 


48  THE  ALBUMINS. 

The  skeletins  are  in  part  related  to  elastin  and  partly  to  collagen. 
Spongin  aud  conchiolin  thus  do  not  give  Millon's  reaction,  and 
accordingly  yield  no  tyrosin  on  decomposition,  while  both  are 
obtained  from  fibroin,  kornein,  and  elastoidin.  It  appears,  on  the 
other  hand,  that,  with  the  possible  exception  of  kornein,  all  the 
skeletins  contain  no  sulphur. 

The  amyloid  substance,  finally,  occupies  a  unique  position  among 
the  albuminoids.  It  is  apparently  met  with  only  under  pathologic 
conditions,  and  is  then  found  in  the  connective  tissues.  Like  the 
true  albumins,  it  consists  of  carbon,  hydrogen,  nitrogen,  oxygen, 
and  sulphur,  and  on  decomposition  yields  both  leucin  and  tyrosin. 
It  gives  Millon's  reaction,  that  of  Adamkiewicz,  and  the  xantho- 
proteic reaction.  It  is  insoluble  in  water,  alcohol,  ether,  dilute 
hydrochloric  acid,  and  acetic  acid.  Concentrated  hydrochloric  acid 
and  solutions  of  the  alkaline  hydrates  cause  its  solution,  but  at  the 
same  time  transform  it  into  acid  albumin  or  alkaline  albuminate. 
The  gastric  juice,  contrary  to  what  has  been  claimed,  likewise  causes 
the  substance  to  dissolve.  Most  characteristic  is  its  behavior  toward 
iodine  and  aniline  green.  The  latter  is  colored  red.  Dilute  aqueous 
solutions  of  iodine  color  the  substance  a  brownish  red  or  a  bluish 
violet,  which  passes  into  blue  on  treating  with  sulphuric  acid.  Iodo- 
methyl  aniline  stains  the  substance  red,  especially  after  previous 
treatment  with  acetic  acid. 

The  more  important  members  of  the  various  groups  which  have 
been  briefly  considered  in  the  preceding  pages,  their  specific  proper- 
ties, and  methods  of  isolation,  will  be  dealt  with  in  greater  detail  in 
connection  with  the  tissues  in  which  they  are  principally  encountered. 
The  derived  albumins  also,  which  are  now  to  occupy  our  attention, 
are  likewise  considered  only  in  a  general  way  at  this  place,  as  we 
shall  have  opportunity  to  study  them  in  greater  detail  in  the 
chapters  on  the  Blood  and  Digestion. 

THE  DERIVED  ALBUMINS. 

Fibrin. — Fibrin  occupies  a  unique  position  among  the  albumins. 
So  far  as  its  general  chemical  composition  goes,  it  is  unquestionably 
closely  related  to  the  albumins  proper.  It  contains  carbon,  hydro- 
gen, nitrogen,  oxygen,  and  sulphur  in  very  much  the  same  propor- 
tion as  the  true  albumins,  and,  like  these,  yields  leucin,  tyrosin, 
glutaminic  acid,  asparaginic  acid,  and  glycocoll  on  decomposition. 
On  the  other  hand,  fibrin  is  insoluble  in  the  common  solvents  of  the 
true  albumins,  viz.,  in  water  and  neutral  saline  solutions.  Acids  and 
alkalies  cause  its  dissolution,  but  during  this  process  the  substance 
itself  is  transformed  into  acid  albumin  or  alkaline  albuminate,  as 
the  case  may  be.  In  this  respect  fibrin  is  closely  related  to  the 
coagulated  albumins.  It  further  merits  a  place  among  the  derived 
albumins,  however,  by  reason  of  its  being  itself  a  derivative  of  a  true 
albumin,  namely,  fibrinogen,  which  is  transformed  into  fibrin  through 


THE  DERIVED  ALBUMINS.  49 

the  agency  of  a  specific  ferment.  Of  the  manner  in  which  this 
transformation  is  effected  we  know  but  little.  According;  to  some 
observers,  the  process  is  essentially  an  oxidation-process.  Others 
maintain  that  the  fibrin  is  present  in  the  fibrinogenous  molecule  in 
combination  with  another  albuminous  group,  and  that  it  results  from 
the  fibrinogen  through  decomposition  of  its  molecule  under  the 
influence  of  the  fibrin-ferment.  However  this  may  be,  it  is  certain 
that  the  fibrinogen  is  not  changed  into  fibrin  through  any  rearrange- 
ment of  its  atoms,  and  that  a  certain  amount  of  another — but 
soluble — proteid,  fibrinoglobulin,  is  obtained  whenever  fibrin  itself 
is  formed.  It  is  hence  a  derivative  of  a  true  albumin,  but  not  a 
native  albumin  itself. 

The  Coagulated  Albumins. — The  coagnlated  albumins  result 
from  the  albumins  proper  through  the  influence  of  heat,  prolonged 
exposure  to  strong  alcohol,  especially  in  the  presence  of  a  neutral 
-alt,  and  in  the  case  of  fibrin  at  least,  which,  as  we  have  just 
seen,  is  closely  related  to  this  group,  through  the  activity  of  a 
specific  ferment.  They  differ  from  the  true  albumins  in  their 
extreme  resistance  to  ail  neutral  solvents,  and  also  to  dilute  acids 
and  alkalies.  Stronger  acids  and  alkalies  cause  their  dissolu- 
tion, with  the  simultaneous  formation  of  acid  albumins  or  alkaline 
albuminates. 

The  Albuminates. — The  albuminates,  as  has  been  pointed  out, 
result  from  the  native  albumins  through  a  process  of  denaturization, 
as  Neunieister  terms  it,  in  consequence  of  which  their  original  char- 
acteristics are  entirely  lost.  Aside  from  their  quantitative  composi- 
tion, they  differ  from  each  other  only  in  so  far  as  they  have  resulted 
through  the  action  of  an  acid  or  an  alkali.  The  alkaline  albu- 
minates thus  contain  less  sulphur  and  less  nitrogen  than  the  acid 
albumins,  as  a  portion  of  the  sulphur  and  the  so-called  amido- 
oitrogen  have  been  split  off.  Both  the  acid  albumins  and  the  albu- 
minates are  insoluble  in  neutral  solvents,  and  are  therefore  precipi- 
tated from  their  solutions  on  neutralization.  They  are  soluble,  on 
the  other  hand,  in  solutions  of  the  alkaline  hydrates,  in  dilute  solu- 
tion- of  sodium  carbonate,  in  hydrochloric  acid,  and  with  a  little 
more  difficulty  in  strong  acetic  acid.  From  their  acid  solutions  they 
are  precipitated  by  salting  with  ammonium  sulphate  or  sodium 
chloride.  Through  the  action  of  an  alkali  acid  albumin  can  be 
transformed  into  alkaline  albuminate,  but  it  is,  of  course,  manifest 

that  tin-  reverse  cannot  occur.  In  the  living  body  the  denaturiza- 
tion of  all  albumins  is  effected  during  the  process  of  digestion,  and 
invariably  precedes  the  formation  of  albumoses  and  peptones. 

The  Albumoses. — The  albumoses  result  from  the  albumins 
proper,  and  also  from  the  albuminoids  and  the  albuminous  radicles 
of  the  proteids,  through  the  action  of  the  so-called  proteolytic  fci-- 
ments,  or  during  their  hydrolytic  decomposition  l>v  means  of  acids 
or  alkalies.  In  every  case  their  formation  is  preceded  by  the 
denaturization  of  the  original  molecule. 
i 


50  THE  ALBUMINS. 

Collectively  the  albumoses  which  are  derived  from  the  true  albu- 
mins, in  contradistinction  to  those  which  are  obtained  from  the 
albuminoids,  are  also  termed  proteoses.  According  to  their  origin, 
we  further  distinguish  between  globulinoses,  vitelloses,  caseoses, 
myosinoses,  keratinoses,  elastoses,  gelatoses,  etc.  One  and  the  same 
albumin,  mqreover,  can  give  rise  to  the  formation  of  different  albu- 
moses. During  the  decomposition  of  fibrin,  for  example,  primary 
albumoses  first  result,  which  are  then  transformed  into  secondary 
albumoses,  and  these  into  the  so-called  peptones.  Formerly  a  dis- 
tinction was  made  between  hemi-albumoses  and  anti-albumoses, 
according  to  the  varying  degree  of  resistance  which  the  individual 
substances  offered  to  the  action  of  trypsin.  While  we  still  recognize 
the  existence  of  hemi-  and  anti-groups  in  the  original  albumins,  it 
appears  from  recent  researches  that  a  complete  separation  of  these 
groups  does  not  occur  at  the  stage  of  digestion  at  which  the  albu- 
moses only  are  found.  These  terms  have  hence  been  abandoned. 
The  primary,  albumoses  were  formerly  also  divided  into  two  groups, 
viz.,  the  proto-albumoses  and  the  hetero-albunioses.  We  now  know, 
however,  that  still  others  are  primarily  formed,  as  will  be  shown 
later.  The  term  dysalbumose  has  been  applied  to  a  variety  of  albu- 
mose  which  apparently  results  from  the  hetero-albumoses  when  these 
are  dried  or  kept  under  water  for  some  time;  dysalbumose  is  then 
insoluble  in  dilute  saline  solution. 

In  their  quantitative  composition  the  albumoses  very  curiously  do 
not  differ  materially  from  the  original  albumins,  and  it  is  hence  dif- 
ficult to  explain  the  relationship  which  exists  between  the  two 
groups.  According  to  most  authorities,  the  albumoses  represent 
hydrolytic  decomposition-products  of  the  albumins,  and  it  has  been 
shown  as  a  matter  of  fact  that  through  the  influence  of  acetic  anhy- 
dride upon  the  so-called  peptones,  which,  as  we  shall  presently  see, 
represent  the  final  products  of  albuminous  digestion,  albuminate-like 
substances  can  be  obtained.  Others,  however,  regard  the  trans- 
formation of  albumins  into  albumoses  as  a  depolymerization  of  the 
original  substance,  while  still  others  look  upon  both  as  isomeric 
bodies. 

The  albumoses  give  the  same  color-reactions  as  their  mother- 
substances.  With  the  biuret  test,  however,  the  original  violet  is 
absent,  and  instead  a  beautiful  rose  color  is  obtained.  Their  final 
decomposition-products  are  the  same  as  those  of  their  antecedents. 

Unlike  the  albumins,  the  albumoses  are  not  entirely  indiffusible, 
and  it  appears  that  the  power  to  pass  through  animal  membranes 
increases  as  they  become  structurally  further  and  further  removed 
from  their  mother-substances. 

As  a  class  the  albumoses  are  much  more  readily  soluble  than  the 
albumins.  Most  of  them  are  soluble  in  water  or  in  dilute  saline 
solutions,  and  also  in  dilute  acids  and  alkalies.  From  their  solu- 
tions they  are  readily  precipitated  by  certain  neutral  salts,  notably 
ammonium  sulphate,  which  precipitates  all  albumoses  when  added  to 


THE  DERIVED  ALB  V MISS.  51 

saturation,  the  reaction  being  slightly  acid.  Most  of  them,  indeed, 
are  thrown  down  when  the  salt  is  added  to  the  extent  of  75 
per  cent.  Each  albumose,  in  fact,  appears  to  possess  certain  special 
limits  of  precipitation  with  ammonium  sulphate  which  enables  ns  to 
separate  the  individual  substances  from  each  other,  and  also  from 
other  albumins  which  may  be  present  at  the  same  time.  Zinc  sul- 
phate behaves  in  a  very  similar  manner.  Sodium  chloride  when 
added  in  substance  to  saturation  causes  a  partial  precipitation  of  the 
albumoses  from  their  neutral  solutions,  while  a  fairly  complete  sepa- 
ration is  obtained  if  to  the  saturated  fluid  is  added  a  small  amount 
of  acetic  acid  that  has  been  saturated  with  the  salt. 

Neutral  or  acid  solutions  of  albumoses  are  not  coagulated  by  heat 
nor  on  treating  with  alcohol,  although  they  are  precipitated  when 
this  is  present  in  considerable  concentration.  After  precipitation, 
however,  they  are  as  soluble  as  before,  and  in  this  respect  they  differ 
very  markedly  from  the  albumins  proper. 

Like  the  native  albumins,  the  albumoses  are  precipitated  by  nitric 
acid,  potassium  ferrocyanide  and  acetic  acid,  metaphosphoric  acid, 
phosphotungstic  acid  in  the  presence  of  hydrochloric  acid,  tannic 
acid,  picric  acid,  trichloracetic  acid,  etc.  ;  but  it  must  be  noted  that 
on  the  subsequent  application  of  heat  the  precipitate  redissolves, 
but  reappears  on  cooling.  The  same  result  is  obtained  by  treating 
a  solution  of  albumoses  with  an  equal  volume  of  a  saturated  solution 
of  sodium  chloride  and  acidifying  with  acetic  acid. 

The  Peptones. — The  term  peptone  is  generally  used  to  designate 
those  final  products  of  albuminous  decomposition  which  result  from 
the  albumoses  on  further  digestion  with  the  proteolytic  ferments,  in 
-'i  far  as  they  still  possess  an  albuminous  character.  According  to 
Kuhne  and  his  school,  they  differ  from  the  albumoses  and  all  other 
albumins  in  not  being  precipitated  from  their  solutions  on  saturation 
with  ammonium  sulphate.  Such  substances  are  obtained  in  abun- 
dance during  the  process  of  tryptic  digestion,  in  vitro  at  least,  while 
during  peptic  digestion  they  are  formed  only  in  small  amounts  and 
on  prolonged  exposure  to  the  ferment. 

I  have  pointed  out  above,  that  Kuhne  distinguishes  between  a 
heini-  and  an  anti-group  in  the  albuminous  molecule,  and  he  accord- 
ingly divide-  the  peptones  into  hemipeptone  and  antipeptone.  Both 
are  supposedly  formed  during  peptic  as  well  as  pancreatic  digestion, 
in  the  Former  instance  the  mixture  of  the  two  substances  is  spoken 
<>f  ,-i-  amphopeptone.     It  is  to  be  noted,  however,  that   hemipeptone 

has  thus  far  not  been  Isolated  as  such,  and  it  appears  indeed  that  the 
substance    ifl    only    theoretically    existent,  and    on    artificial    digestion 

with  trypsin  i-  further  decomposed  into  amido-acids,  while  in  the 
digestive  trad  it  is  supposedly  taken  up  at  once  by  the  epithelial 
cells  ami  transformed  into  albumin  proper.  Antipeptone,  on  the 
other  hand,  to  judge  from  recent  investigations,  represents  a  mixture 
of  acid  and  basic  substances,  among  which  Latter  the  so-called  hexon 

DflSdfl    are    found.       It   is  thus   dear  that  with    the   possible   exception 


52  THE  ALBUMINS. 

of  the  theoretical  hem i peptone,  we  are  not  warranted  in  speaking  of 
peptones  as  a  well-defined  chemical  unity,  and  it  is  questionable, 
indeed,  whether  the  final  products  of  proteolytic  digestion  can 
actually  be  classed  as  albumins.  We  shall  accordingly  not  give  an 
account  of  the  various  properties  of  the  peptones  which  would  be 
based  upon  the  conception  that  we  are  dealing  with  a  well-defined 
chemical  substance.  We  shall  have  occasion,  however,  at  another 
place  to  deal  in  detail  with  some  of  the  better  known  constituents 
of  antipeptone. 


CHAPTER     III. 

THE  CARBOHYDRATES. 

It  has  been  pointed  out  in  the  preceding  chapter  that  while 
plants  are  capable  of  effecting  from  relatively  simple  compounds  the 
svnthesis  of  those  complex  albumins  which  are  found  in  their 
various  tissues  and  organs,  animals  do  not  possess  this  power, 
and  are  therefore  dependent  for  their  supply  of  nitrogen  upon  the 
albuminous  food-stuff's  that  have  been  elaborated  by  plants.  The 
carbohydrate  supply  of  animals  is  also  derived  from  plants,  but  for 
the  maintenance  of  life  it  is  not  necessary  that  the  carbohydrates 
should  be  furnished  as  such,  as  animals  are  not  only  capable  of 
splitting  off  the  carbohydrate  radicle  of  the  albuminous  molecule  as 
occasion  demands,  but,  as  we  shall  see  later,  they  can  also  form  carbo- 
hydrates directly  from  the  fats  which  are  stored  in  their  tissues.  The 
carbohydrates  cannot  therefore  be  regarded  as  essential  food-stuffs, 
and  we  see,  as  a  matter  of  fact,  that  carnivorous  animals,  at  least, 
are  capable  of  existing  on  albuminous  food  exclusively.  Carbo- 
hydrates are  important,  however,  as  the  stored  energy  which  is  thus 
supplied  to  animals  represents  a  considerable  caloric  value,  and  they 
can  hence  protect  the  albumins  from  undue  destruction.  The  im- 
portance of  the  carbohydrates  as  food-stuffs  is  thus  only  secondary, 
and  they  are  totally  unable  to  take  the  place  of  the  albumins.  All 
living  matter  requires  a  definite  amount  of  nitrogen  so  that  life  may 
be  maintained,  and  if  this  is  withdrawn  death  inevitably  results. 
It  is  to  be  noted,  however,  that  whereas  animals  can  exist  without 
carbohydrate  food,  and  whereas  the  albumins  largely  predominate  in 
ii-  (issues,  the  reverse  holds  good  for  plants.  Here  the  carbo- 
hydrates prevail,  while  the  albumins  are  much  less  abundant.  Con- 
sequently we  may  expect  to  find  a  far  greater  diversity  of  carbo- 
hydrate-, in  the  vegetable  than  in  the  animal  world.  This  is 
actually  the  case.  Ajb  it  would  lead  too  far,  in  a  work  of  this 
-'•ope,  however,  to  consider  all    those  carbohydrates    which   occur   in 

the  vegetable  world,  we  -hall  confine  our  attention  in  the  subsequent 
pagi  -  10  those  form-  which  may  be  regarded  as  common  food-stuffs, 

OT  those  which  are  more  or  Less  peculiar  to  the  animal  body. 

All  carbohydrates  consisl  of  carbon,  hydrogen,  and   oxygen,  and 

in  mo-t  members  of  the  group  tin'   elements    hydrogen    and    oxygen 

are  presenf  in  such  proportion  a-  to  form  water,  [n  others,  how- 
ever, this  i-  not  the  case;  and  there  are  substances,  such  as  lactic 
acid  .Mid  acetic  acid  which  likewise  contain  hydrogen  and  oxygen  in 

this    proportion,  but    which    are    manifestly    not   carbohydrates.      As 


54  THE  CARBOHYDRATES. 

there  are  no  specific  properties  peculiar  to  these  substances  as  a 
class,  it  is  impossible  to  give  an  adequate  definition  of  what  is  meant 
by  the  term  carbohydrate.  Chemically  speaking,  they  are  deriva- 
tives of  polyatomic  alcohols,  and  of  the  nature  of  aldehydes  or 
ketones.  They  are  conveniently  divided  into  monosaccharides, 
disaccharides,  and  polysaccharides.  The  disaccharides  and  poly- 
saccharides differ  from  the  monosaccharides  in  being  more  complex 
substances  and  apparently  built  up  from  the  monosaccharides 
through  a  condensation  of  monosaccharine  anhydrides  to  form  a 
double  or  a  multiple  group.  Accordingly,  on  hydrolytic  decom- 
position they  yield  two  or  more  monosaccharine  molecules  for 
every  original  molecule,  as  is  shown  below : 

(1)  C6H1206  —   glucose,  viz.,  leevulose. 

(2)  C12H22On    +   H20  =  CfiH1206   +   C6rl]206. 
Cane-sugar  Glucose.  Lsevulose. 

(Disaccharide.)  (Monosaccharides.) 

THE  MONOSACCHARIDES. 

According  to  the  number  of  carbon  atoms  which  are  present  in 
the  molecule,  the  monosaccharides  can  be  divided  into  trioses,  tet- 
roses,  pentoses,  hexoses,  heptoses,  octoses,  etc.  Of  these,  the  hex- 
oses  only  will  be  considered,  as  the  remaining  groups  are  of  practi- 
cally no  significance  as  animal  food-stuffs,  and  are  in  man,  at  least, 
mostly  eliminated  through  the  kidneys  as  foreign  matter. 

The  most  important  representatives  of  the  hexoses  are  glucose,, 
which  is  also  termed  dextrose ;  lsevulose  or  fructose ;  mannose  and 
galactose.  Some  of  these,  such  as  glucose  and  lsevulose,  are  found 
free  in  nature,  or  they  result  as  hydrolytic  decomposition-products 
from  the  more  complex  carbohydrates  and  related  nitrogenous  sub- 
stances, the  so-called  glucosides.  They  are  all  derivatives  of  the 
stereo-isomeric  hexatomic  alcohols  of  the  composition  CH2.OH — 
(CH.OH)4 — CH2.OH.  Of  these,  three  are  known  to  occur  in  the 
natural  state,  viz.,  sorbite,  or  glucite,  mannite,  and  dulcite.  As  has 
been  pointed  out  above,  the  monosaccharides  are  either  aldehydes  or 
ketones,  and  we  accordingly  find  that  glucose,  mannose,  and  galac- 
tose represent  the  aldehydes  (aldoses)  of  sorbite,  mannite,  and 
dulcite,  respectively,  while  lsevulose  is  the  ketone  (ketose)  of 
mannite.  They  can  therefore  be  represented  by  the  structural 
formulse  : 

(1)  CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO  (glucose,  mannose,  and 

galactose). 

(2)  CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH)  (kevulose). 

As  a  matter  of  fact  it  is  possible  to  transform  these  hexoses  into 
their  corresponding  alcohols  by  careful  reduction,  and  vice  versa. 

In  accordance  with  their  character  as  aldehydes  or  ketones,  the 
aldoses  on  oxidation  yield  oxyacids,  which  have  the  same  number 


THE  MONOSACCHARIDES.  55 

of  carbon  atoms  as  the  original  substances,  while  the  ketoses  give 
rise  to  acids  which  have  a  smaller  number  of  carbon  atoms.  The 
oxyacids  which  arc  derived  from  the  aldoses,  moreover,  are  either 
monobasic  or  dibasic,  according  to  the  extent  to  which  the  oxida- 
tion has  been  carried. 

These  changes  are  represented  by  the  equations  : 

(1)  CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO  +  O  = 

Glucose.  CHa(OH).(CH.OH  )4.COOH. 

Gluconic  acid. 

(2)  CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO  +  30  = 

Glucose.  COOH.(CH.OH)4.COOH  +  H,0. 

Saccharinic  acid. 

The  acids  which  can  thus  be  obtained  from  the  aldoses  glucose, 
mannose,  and  galactose,  are  the  monobasic  acids — gluconic,  man- 
nonie,  and  galactonic  acid ;  and  the  dibasic  acids — saccharinic,  man- 
nosaccharinic,  and  mucinic  acid.  Of  these,  saccharinic  acid  is  of 
special  interest,  as  it  can  readily  be  transformed  into  saccharolactonic 
acid,  which  in  turn  yields  glucuronic  acid.  This  latter,  as  we  shall 
see  later,  is  found  also  in  the  animal  body.  It  is  an  aldehydic  acid, 
and  stands  midway  between  gluconic  acid  and  saccharinic  acid.  It 
is  represented  bv  the  formula  COOH.CH(OH).CH(OH).CH(OH). 
CH(OH).COH. 

The  hexoses  are  colorless  and  odorless  substances  of  a  sweetish 
taste  ;  they  present  a  neutral  reaction,  and  are  readily  soluble  in 
water,  with  difficulty  in  absolute  alcohol,  and  insoluble  in  ether. 
They  can  be  obtained  in  crystalline  form,  and  diffuse  through  animal 
membranes.  Some  of  them  are  dextrorotatory,  others  laevorotatory, 
while  still  others  are  optically  inactive.  They  are  strong  reducing- 
substances,  and  for  the  most  part  fermentable  with  yeast.  Espe- 
cially interesting  further  is  the  behavior  of  the  hexoses  toward  the 
hydrazins  in  the  presence  of  acetic  acid,  with  which  they  form 
hydrazo7t8.  These  can  be  further  transformed  into  osazons,  which 
arc  very  characteristic  substances,  and  may  serve  to  distinguish  the 
various  sugars  from  each  other.  On  decomposition  with  fuming 
hydrochloric  acid  the  osazons  then  give  rise  to  the  formation  of 
ogon8 — /'.'•.,  keto-aldehydes,  which  can  be  further  reduced  to  ketoses. 
By  starting  with  an  aldose,  it  is  thus  possible  to  obtain  an  isomeric 
ketose.     These  changes  may  be  represented  by  the  equations: 

I     CH,(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO   •   <  •;H,i.xir.\IT,= 
Glucose  Phenylhydrazin. 

CH,(OH).CH(OH).CH(OH  |.CH(0H).CH(0H).CH:N.NH.C6H6    H,0 
Phony  lglucohydrazon. 

■  II    OH  M  II  "II  .<  II  "II    I  II  on  ,CH(OH).CH:N.NH.CaH6   | 

<;n  ..\n.Nir!l  = 
CH,(OH    CH(OH).CH(OH).CH(OH  |.C.CH.N.NH.CeH,  |-  H2<>  +  211. 
Phenylglucosazon. 

ssmru6. 


56  THE  CARBOHYDRATES. 

(3)  CH2(0H).CH(0H).CH.(0H).CH(0H).C.CHN.NH.C6H5+2H30+2HC1  = 

N.NHC6H5. 
2NH3.NH.C6H5.HCl  +  CH2(OH).CH(OH).CH(OH).CH(OH).CO.COH. 

Oson. 

(4)  CH2(OH).CH(OH).CH(OH).CH.(OH).CO.CHO  +  2H= 

CH2(OH).  [CH(OH)]3.COCH2(OH) 

Lsevulose. 

The  same  result  may  be  reached  when  the  corresponding  osazon 
of  the  aldose  is  directly  reduced,  and  the  resulting  osamin  is  treated 
with  nitrous  acid.  The  glucosamin  thus  obtained  as  an  intermediary 
product  is  of  special  interest  in  so  far  as  it  also  results  from  the  de- 
composition of  the  hyalins  chitin  and  chondroitin.  By  oxidation  with 
bromine  glucosamin  then  yields  chitonic  acid,  from  which  the  cor- 
responding sugar,  chitose,  can  be  obtained  on  reduction.  The 
changes  which  are  here  involved  may  be  represented  by  the  equa- 
tions : 

(1)  CH2(OH).CH(OH).CH(OH).CH(OH).C.CH.N.NH.C6H6  +  H20  +  4H  = 

Phenylglucosazon 

N.NH.C6H5 
CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(NH,)  + 

Glucosamin  C6H5.NH2.NH  +  C6HB.NH2 

Phenylhydrazin  anilin 

(2)  CH2(OH).CH(OH).CH(OH.)CH(OH)CO.CH2(NH2)  +  HN02  = 

Glucosamin 
H20  +  2N  +  CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH) 

Leevulose. 

(1)  C,sH,0N2O12  +  4H20==2CH2(OH).CH(OH).CH(OH):CH(OH). 

Chitin.  '     Glucosamin.  CO.CH2(NH2)   +  3CH3.COOH 

(2)  CH.,(OH).CH(OH).CH(OH.)CH(OH).CO.CH2(NH2)  +  40  = 

CH2(OH).CH(OH).CH(OH).CH.(OH).CH(OH).COOH  +  HN02 
Chitonic  acid 

(3)  CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).COOH  +  2H  = 

CH2(OH).[CH(OH)]4.COH  +  H20 
Chitose 

On  boiling  with  dilute  mineral  acids  the  hexoses  are  decomposed 
into  formic  acid,  lsevulinic  acid,  and  certain  humin  substances.  With 
the  alkalies,  on  the  other  hand,  they  yield,  besides  other  products, 
also  Isevulinic  acid  and  a  ketonic  acid  of  the  composition  CH3.CO. 
CH2.CH2.COOH.  On  the  application  of  dry  heat  they  form  so- 
called  caramel,  and  are  finally  carbonized. 

As  stated  above,  most  of  "the  hexoses  are  capable  of  undergoing 
fermentation — i.  e.,  a  decomposition  which  is  effected  through  the 
activity  of  certain  minute  organisms.  According  to  the  character 
of  the  specific  organism  present,  we  distinguish  between  alcoholic 
and  acid  fermentation,  such  as  lactic  acid,  butyric  acid,  and  acetic 
acid  fermentation.  The  former  is  brought  about  through  the  influ- 
ence of  various  varieties  of  yeast,  while  the  latter  are  referable  to  the 
activity  of  certain  bacteria,  such  as  the  Bacterium  lactis  aerogenes, 
the  Bacillus  acidi  butyrici,  the  Mycoderma  aceti,  etc.     The  decom- 


THE  DISACOHABIDES.  57 

positions  which  are  thus  effected  may  be  represented  by  the  equa- 
tions : 

(1)  CeH^Og  =  2C,II5(OH)  +  C02. 

Ethyl  alcohol. 

(2)  C6H1206  =  2CH3— CH.OH-COOH. 

Lactic  acid. 

(3)  2C3H603  =  C3HT.COOH       2COa  +  4H. 

Lactic  acid.       Butyric  acid. 

Of  the  hexoses,  glucose  only  is  found  in  the  animal  body  ;  while 
laevulose,  mannose,  and  galactose  do  not  occur  as  such,  and  on  reach- 
ing the  liver  are  apparently  immediately  transformed,  together  with 
glucose,  into  the  polysaccharide  glycogen.  Whether  or  not  a  trans- 
formation into  glucose  first  takes  place,  and  whether  this  can  occur 
in  the  intestinal  mucosa,  is  unknown.  The  amount  of  glucose  which 
may  be  found  in  the  blood  and  lymph,  and  in  the  various  tissues  of 
the  body,  is  always  small. 

Laevulose  occurs  in  nature  together  with  glucose,  most  abundantly 
in  various  fruits,  the  roots  and  seeds  of  many  vegetables,  and  also  in 
honey.  It  further  results  during  the  hydrolytic  decomposition  of 
cane-sugar,  inulin,  and  other  carbohydrates.  It  is  readily  soluble  in 
water,  and  its  aqueous  solutions,  in  contradistinction  to  common 
glucose,  are  hevorotatory.  It  may  be  obtained  in  crystalline  form, 
but  with  difficulty.  It  is  fermentable,  and  gives  the  same  reduc- 
tion-tests as  glucose  (which  see).  With  phenylhydrazin  laevulose 
yields  the  same  osazon. 

Galactose  is  formed  during  the  hydrolytic  decomposition  of 
lactose  and  many  other  carbohydrates.  It  is  also  obtained  from 
eerebrin  on  heating  with  dilute  mineral  acids.  It  is  not  so  readily 
soluble  in  water  as  glucose,  but  like  it  is  dextrorotatory.  Galactose 
crystallizes  in  needles  and  platelets  which  melt  at  168°  C.  It  is 
fermentable,  and  yield-  an  osazon  which  melts  at  193°  C.  It  re- 
duce- an  alkaline  solution  of  cupric  oxide,  but  to  a  less  marked 
degree  than  glucose.  On  oxidation  it  yields  first  galactonic  add  and 
later  mucinic  arid. 

Glucose  will  be  considered  in  a  subsequent  chapter,  where  the 
methods  of  testing  for  the  simple  sugars  in  general,  and  also  their 
quantitative  estimation,  will  be  described. 

THE    DISACCHARIDES. 

The  disaccharidea  result  from  the  monosaccharides  through  a  con- 
densation of  the  anhydrides  of  two  monosaccharine  molecules,  analo- 
gous to  the  formation  of* ethers  from  alcohol-.  On  hydrolytic  de- 
composition they  accordingly  yield  two  monosaccharine  molecules, 
which  represent  either  one  ana  the  same  substance  or  two  isomeric 
bodies.  Some  of  the  disaccharidea  occur  in  nature  as  such,  while 
other-  result  from  the  decomposition  of  still  more  complex  carbo- 
hydrates. 


58  THE  CARBOHYDRATES. 

The  most  important  members  of  the  group  are  cane-sugar  or 
saccharose,  lactose,  maltose,  and  isomaltose.  They  are  all  hexo- . 
bioses — i.  e.,  they  represent  the  union  of  the  anhydrides  of  two 
hexoses,  and  can  therefore  be  represented  by  the  general  formula 
C12H22Ou.  Of  these,  cane-sugar  is  formed  through  the  union  of 
one  molecule  of  glucose  and  one  molecule  of  lsevulose  ;  lactose  from 
glucose  and  galactose;  while  maltose  contains  two  molecules  of 
glucose. 

In  their  general  properties  the  disaccharides  closely  resemble  the 
monosaccharides.  Like  these,  they  have  a  sweet  taste.  They  are 
crystallizable,  capable  of  passing  through  animal  membranes,  and 
are  optically  active.  In  certain  particulars,  however,  differences 
exist.  Lactose,  maltose,  and  isomaltose  are  thus  capable  of  reducing 
metallic  oxides  in  alkaline  solution,  and  yield  osazons  with  phenyl- 
hydrazin,  while  saccharose  does  not  react  in  this  manner. 

The  disaccharides  as  such  are  not  fermentable,  but  only  after  inver- 
sion to  monosaccharides.  It  is  true  that  a  solution  of  cane-sugar  or 
of  lactose  will  undergo  alcoholic  fermentation  when  exposed  to  the 
action  of  yeast;  but  we  now  know  that  the  yeast-cell  is  capable  of 
furnishing  certain  ferments  which  belong  to  the  class  of  the  so-called 
non-organized  ferments,  and  which  themselves  are  capable  of  bring- 
ing about  the  inversion  of  these  more  complex  carbohydrates.. 
Emil  Fischer,  moreover,  has  shown  that  for  the  inversion  of  a 
special  disaccharide  a  specific  ferment  is  necessary.  As  the  various 
fermentative  agents,  however,  possess  a  varying  number  of  inverting 
ferments,  it  is  clear  that  a  certain  disaccharide  may  be  inverted  by 
one  form  of  yeast,  but  not  by  another ;  while,  on  the  other  hand, 
one  special  form  may  be  capable  of  inverting  all  forms  of  the 
disaccharides.  This  is  actually  the  case,  and  we  thus  find  that  the 
so-called  kefir  granules,  as  also  the  Bacterium  lactis,  can  invert  cane- 
sugar  as  well  as  maltose  and  lactose.  Common  yeast,  on  the  other 
hand,  inverts  only  cane  sugar  and  maltose,  while  lactose  is  not 
attacked. 

According  to  their  specific  power  of  inversion,  these  ferments  are 
spoken  of  as  invertin,  maltase,  and  lactase.  The  derivation  of  the 
two  latter  names  is,  of  course,  apparent.  The  significance  of  the 
term  invertin,  however,  is  not  so  clear.  It  has  reference  to  the 
mixture  of  glucose  and  lsevulose  which  is  obtained  from  cane-sugar 
by  inversion,  and  which  was  originally  spoken  of  as  invert-sugar. 
Invertin  is  thus  a  ferment  which  inverts  cane-sugar. 

After  inversion  the  disaccharides  undergo  fermentation,  like  the 
monosaccharides,  and  here,  as  there,  we  may  distinguish  between 
alcoholic,  lactic  acid,  butyric  acid,  and  acetic  acid  fermentation. 

Cane-sugar  is  found  in  nature  most  abundantly  in  sugar-cane,  in 
the  roots  of  the  sugar  beet,  and  in  the  stems  of  certain  plants. 
The  pure  substance  is  crystalline,  and  melts  at  160°  C.  On  further 
heating  it  turns  brown  and  forms  so-called  caramel.  It  is  easily 
soluble   in  water,  and  turns  the  plane  of  polarization  to  the  right. 


THE  POLYSACCHARIDES.  59 

As  stated,  it  does  not  yield  an  osazon  and  does  not  reduce  metal- 
lic oxides.  After  inversion  with  invertin  it  undergoes  the  same 
fermentations  as  the  resulting  monosaccharides.  On  oxidation 
it  vields,  in  addition  to  other  substances,  saccharinic  and  oxalic 
acids. 

Maltose  does  not  occur  in  nature  as  such,  but  results  during  the 
digestion  of  starch  and  glycogen  in  the  alimentary  canal.  It  is  a 
crystalline  substance,  which  is  easily  soluble  in  water  and  turns  the 
plane  of  polarization  to  the  right.  With  phenylhydrazin  it  yields 
an  osazon — maltosazon — which  melts  at  206°  C.  It  readily  under- 
goes fermentation,  and  like  glucose  reduces  metallic  oxides  in  alka- 
line solution,  but  to  a  less  degree. 

Isomaltose  results  from  starch  through  the  action  of  a  diastatic 
ferment.  In  the  intestinal  canal  it  is  thus  found  together  with 
maltose.  It  is  easily  soluble  in  water  and  turns  the  plane  of  polari- 
zation to  the  right.  Its  osazon  melts  at  150°  to  153°  C.  It  under- 
goes fermentation,  but  much  more  slowly  than  maltose. 

Lactose,  which  is  almost  exclusively  found  in  the  animal  world, 
will  be  considered  in  a  subsequent  chapter  (see  Milk). 

THE    POLYSACCHARIDES. 

The  polysaccharides  result  from  the  monosaccharides  in  the  same 
wav  as  the  disaccharides.  In  other  words,  they  represent  the 
anhydrides  of  the  monosaccharides,  of  which  many  molecules,  how- 
ever, are  condensed  to  form  the  resulting  polysaccharine  molecule. 
Their  general  formula  therefore  is  (C6H1(JOr))x,  in  which  x  is  a  vari- 
able factor,  but  exceeds  two.  In  many  cases  the  value  of  x  is 
unknown,  but  it  is  probably  always  large.  From  a  determination 
of  the  -izc  of  the  starch  molecule,  for  example,  we  may  conclude 
that  x  in  this  case  is  equivalent  to  108.  In  others,  such  as  glycogen 
and  the  dextrins,  however,  it  is  certainly  much  smaller.  In  con- 
formity with  their  structure,  the  polysaccharides  all  yield  mono- 
saccharides on  hydrolytic  decomposition.  During  this  process, 
however,  a  variable  number  of  intermediary  products  are  formed, 
which  may  themselves  be  polysaccharides,  though  of  a  lower  order, 
and  which  in  turn  yield  disaccharides  and  finally  monosaccharides. 
Starch  is  thus  first  transformed  into  erythrodextrin,  which  in  turn 

yield-  achroodextrin  ;  this  is  further  changed  to  isomaltose,  and  then 
to  maltose,  which  finally  yields  glucose.  In  other  eases,  ;is  with 
glycogen,  the  disaccharides  isomaltose  and  maltose  are  formed 
directly.  Cellulose  likewise  yields  glucose  as  a,  final  product,  while 
Uevulose  results  from  inulin,  and  mannose  from  the  so-called  reserve 
celluloses,  which  are  found  in  the  cell-walls  of  many  seeds.  Galac- 
tose i-  similarly  obtained  from  many  gums,  and  from  a  variety 
of  cellulose,  which  Schultze  hae  termed  galactose-cellulose,  in  con- 
tradistinction to  the  mannose-cellulose  and  the  true  dextrose-cellu- 
loses.     J 1 1  many  instances,  however,  the  exaci   mode  of  decomposi- 


60  THE  CARBOHYDRATES. 

tion,  as  also  the  character  and  number  of  the  intermediary  products, 
is  but  imperfectly  understood. 

In  their  physical  and  chemical  properties  considerable  differences 
exist  between  the  polysaccharides  and  the  other  carbohydrates 
which  have  so  far  been  described.  They  are  thus  non-crystal lizable 
substances  and  devoid  of  a  sweet  taste.  In  alcohol  and  ether  they 
are  insoluble.  In  water  most  of  them  are  more  or  less  soluble,  but 
as  a  class  they  are  incapable  of  diffusing  through  animal  membranes. 
From  their  solutions  they  can  be  precipitated  by  saturation  with 
neutral  salts,  and  notably  with  ammonium  sulphate.  Like  the 
monosaccharides  and  disaccharides,  they  are  optically  active,  but 
with  the  exception  of  the  dextrins  they  do  not  reduce  metallic 
oxides  in  alkaline  solution,  and  none  of  them  combine  with  phenyl- 
hydrazin  to  form  osazons.  As  such,  they  are  incapable  of  under- 
going fermentation  ;  but,  like  the  disaccharides,  they  may  be  inverted 
to  monosaccharides  through  various  ferments  or  acids,  and  can  then 
be  further  decomposed. 

Especially  important  is  their  behavior  toward  iodine,  with  which 
most  of  the  polysaccharides  combine  to  form  colored  compounds  that 
are  quite  characteristic.  Starch  is  thus  colored  blue,  glycogen  a 
mahogany  brown,  and  erythrodextrin  red. 

The  polysaccharides  which  are  used  as  food-stuffs  are  conveni- 
ently divided  into  starches,  vegetable  gums  and  dextrins,  and  cellu- 
loses. Of  these,  the  starches  are  by  far  the  most  important,  as  they 
include  not  only  vegetable  starches  and  glycogen,  but  also  give  rise 
to  formation  of  the  dextrins. 

Like  the  disaccharides,  all  these  substances  finally  give  rise  to  the 
formation  of  glycogen,  but  it  appears  that  they  are  previously  trans- 
formed into  glucose,  and  that  this  transformation  takes  place  in  the 
epithelial  lining  of  the  intestinal  canal. 

Starch  occurs  widely  distributed  in  the  vegetable  world,  and  con- 
stitutes the  most  important  reserve  food  of  most  of  the  higher 
plants.  It  is  found  in  the  form  of  distinct  granules,  which,  on 
microscopic  examination  exhibit  a  marked  concentric  striation,  and 
which  differ  in  size  and  form  in  different  plants.  The  individual 
granules  are  enclosed  in  a  capsule  of  so-called  starch  cellulose,  which 
is  insoluble  in  water,  but  which  can  be  made  to  open  by  heating  in 
the  presence  of  much  water.  The  contained  starch -prarm  fo.se  can 
thus  be  obtained,  and  constitutes  the  so-called  soluble  starch,  amylum 
or  amylodextrin.  During  this  process  no  doubt  a  still  more  complex 
molecular  group  of  monosaccharine  anhydrides  is  decomposed,  but 
of  the  intermediary  products  which  are  thereby  formed,  if  any, 
nothing  is  known.  In  the  alimentary  canal  this  change  is  effected 
through  the  activity  of  certain  ferments,  which  then  further  give 
rise  to  the  formation  of  dextrin,  maltose,  isomaltose,  and  a  certain 
amount  of  glucose.  On  boiling  with  dilute  acids  glucose  is  formed, 
with  various  dextrins  as  intermediary  products.  Among  these,  as 
has  been   shown,  erythrodextrin  is  apparently  the  first  to  develop, 


THE  POLYSACCHARIDES.  61 

achroodextrin  appears  later,  and  from  this  isomaltose,  maltose,  and 
glucose  are  finally  obtained.  It  appears,  however,  that  during  the 
decomposition  of  achroodextrin  at  least  still  other  dextrins  of  lower 
molecular  weight  are  simultaneously  formed,  which  in  turn  yield 
maltose  and  glucose.  But  finally  one  dextrin  is  obtained  which 
undergoes  no  further  change,  and  which  is  termed  maltodextrin. 

Most  characteristic  is  the  behavior  of  starch  toward  iodine,  with 
which  it  gives  an  intense  blue  color  that  disappears  on  heating,  but 
reappears  on  cooling.  In  a  solution  of  sodium  or  potassium  hydrate 
starch  swells  up  and  forms  a  paste. 

Inulin  and  lichenin,  which  also  belong  to  the  starches,  and  which 
occur  in  the  roots  of  various  composites  and  in  lichens,  respectively, 
are  insignificant  as  food-stuffs  and  need  not  be  considered. 

Glycogen,  which  likewise  belongs  to  this  class,  and  is  known  also 
as  animal  starch,  is  largely  formed  in  the  animal  body  and  represents 
one  of  its  most  important  constituents.  It  will  be  considered  in 
detail  in  a  subsequent  chapter. 

Dextrins. — The  dextrins,  as  has  been  shown,  are  formed  from 
starch  during  its  hydrolytic  decomposition  by  means  of  ferments  or 
on  boiling  with  dilute  mineral  acids.  To  a  certain  extent  they 
result  also  when  starch  is  heated  to  a  temperature  of  from  200° 
to  210°  C.  Through  continued  decomposition  they  give  rise  to 
maltose  and  isomaltose,  and  finally  to  glucose. 

As  a  class  the  dextrins  are  easily  soluble  in  water  and  turn  the 
plane  of  polarization  to  the  right.  From  the  other  polysaccharides 
they  differ  in  their  ability  to  dissolve  cupric  hydroxide  in  alkaline 
solution.  With  erythrodextrin  iodine  strikes  a  red  color,  while 
achroodextrin  is  unaffected. 

The  so-called  vegetable  gums  and  vegetable  mucins  will  not  be 
considered,  as  they  are  of  no  importance  as  food-stuff's.  To  this 
class  belong  the  so-called  gum  Arabic,  wood  gum,  cherry  gum,  etc., 
as  also  the  various  pectins. 

Celluloses. — As  food-stuffs  the  celluloses  are  likewise  of  second- 
arv  importance.  They  arc  considered,  however,  at  this  place  owing 
to  their  wide  distribution  in  the  vegetable  world,  where  they  form 
the  greater  portion  of  all  cell-envelopes.  In  the  animal  world 
they  are  Likewise  encountered,  and  enter  largely  into  the  composi- 
tion of  tlic  external  skeleton  of  the  tunicates,  (lie  arthropods,  and 
Bome  of  tic  cephalopoda.  They  arc  characterized  by  their  extreme 
resistance  to  the  mosl  diver-  solvents,  and  arc  indeed  soluble  only  in 
a   solution    of  CUpric    hydroxide    in    Strong   ammonia — the    so-called 

Schweitzer's  reagent.  From  this  solution  the  substance  can  !><■ 
obtained  in  amorphous  form  on  precipitation  with  acids.  Moder- 
ately concentrated  sulphuric  acid  transforms  cellulose  into  vegetable 
amyloid,  which  i-  colored  blue  by  iodine.  With  concentrated  nitric 
acid,  or  with  ;i  mixture  of  nitric  acid  and  concentrated  sulphuric 
acid,  it  yields  the  highly  explosive  nitrocelluloses. 

Wood  (lignin)and  cork  arc  derivatives  of  cellulose.     On  hydro- 


62  THE   CARBOHYDRATES. 

lytic  decomposition  the  common  cellulose  yields  glucose,  while  the 
so-called  hemicelluloses  give  rise  to  galactose  or  rnannose,  as  also  to 
certain  pentoses,  such  as  arabinose  and  xylose.  In  the  intestinal 
canal  a  certain  portion  of  the  ingested  cellulose  is  unquestionably 
dissolved.  The  products,  however,  to  which  it  gives  rise  are  for  the 
most  part  unknown.  Certain  micro-organisms  which  are  present  at 
the  time  bring  about  a  fermentation  of  the  substance,  during  which 
marsh  gas,  acetic  acid,  and  butyric  acid  are  formed,  but  the  greater 
portion  no  doubt  is  eliminated  in  the  feces  as  such. 


CHAPTER     IV. 

THE  FATS. 

The  origin  of  fats  in  the  animal  body  is  threefold  :  one  portion  is 
derived  from  the  fats  which  have  been  ingested  as  food ;  another 
portion  is  formed  from  the  carbohydrates ;  while  a  third  portion 
results  from  the  decomposition  of  albumins.  As  food-stuffs  the  fats 
are  of  great  importance,  because  their  caloric  value  is  quite  high — 
higher  in  fact  than  that  of  the  carbohydrates  and  albumins ;  but, 
like  the  carbohydrates,  they  are  unable  to  take  the  place  of  the 
albumins.  Animals  that  are  fed  exclusively  on  fats  die  sooner  or 
later,  although  they  may  become  quite  fat  during  the  period  of  their 
special  diet.  In  the  animal  body  they  represent  a  variable  amount 
of  reserve  food,  which  is  conveniently  stored  in  the  subcutaneous 
areolar  tissue,  in  the  omentum  and  mesentery,  in  the  bone-marrow, 
etc.  In  case  of  inanition  it  is  utilized  long  before  the  tissues  of  the 
body  proper  are  attacked,  and  we  accordingly  find  that  in  persons 
who  have  died  from  wasting  diseases  every  vestige  of  fat  may  have 
disappeared,  while  the  muscular  nutrition  may  still  be  fair. 

That  portion  of  the  body  fat  which  is  derived  from  the  fats 
ingested  as  such  is  really  the  smallest  portion,  and  by  far  the  greater 
amount  results  from  the  carbohydrates.  The  manner  in  which  this 
transformation  takes  place  is  unknown,  but  it  is  probable  that  the 
carbohydrates  which  are  utilized  for  this  purpose  are  first  decom- 
posed, and  then  reduced  ;  and  that  the  fats  finally  result  through 
a  synthesis  of  Buch  redaction-products.  Such  syntheses,  however, 
cannot  at  once  be  compared  to  those  which  take  place  in  plants,  for 
here  we  have  <o<-n  that  the  fats  can  be  formed  directly  from  water 
and  carbon  dioxide.  In  animals  this  does  not  occur,  and  we  can 
definitely  state  that  if  the  fats  are  formed  in  the  manner  indicated 
at  all,  they  resull  from  very  much  more  complex  molecules. 

The  origin  of  the  fats  from  carbohydrates  can  be  demonstrated  in 
various  ways.  Dumas  and  W.  Milne  Edwards  have  shown  that 
bees  which  are  fed  exclusively  on  sugar  produce  three  times  as  much 
wax  a-  compared  with  that  which  was  originally  present  in  their 
bodies.  It  i-  a  well-known  feet,  moreover,  that  cattle  which  arc  fed 
on  nitrogenous  food  exclusively  do  not  fatten,  or  only  slightly  so; 
whereas    they    BOOH    Lrain    in    weight    when    a    certain    proportion  of 

carbohydrates  is  added  to  their  food. 

The'  proportion  of  fit  which  is  normally  derived  from  albumins 
is  not  very  large,  if  we  except  the  period  of  lactation  in  female 
animals,   bat    it-    possible   origin    from    this   source  is  undoubted. 

83 


64  THE  FATS. 

Bitches  which  are  fed  solely  on  lean  meats  continue  to  furnish  milk 
containing  an  abundance  of  butter.  Pettenkoffer  and  Yoit  further 
showed  in  dogs  that  when  the  carbohydrates  remained  constant,  but 
the  albuminous  food  was  increased,  a  steady  gain  in  fat  occurred,  'as- 
shown  in  the  table : 

Carbohydrates 

ingested.  Meat  ingested.  Gain  in  fat. 

379  grams.  211  grams.  24  grams. 

379      "  608      "  55      " 

379      "  1469      "  112      " 

A  further  illustration  is  had  in  the  transformation  of  the  muscular 
tissue  of  cadavers  into  so-called  adipocere,  a  substance  which  con- 
sists to  the  extent  of  97  per  cent,  of  ammonium  palmitate  with  a 
small  amount  of  stearate. 

Under  various  pathologic  conditions,  finally,  we  can  follow  with 
the  microscope  the  gradual  transformation  of  albuminous  material 
into  fat. 

All  fats  consist  of  carbon,  hydrogen,  and  oxygen.  They  are 
insoluble  in  water,  slightly  soluble  in  cold  alcohol,  while  in  hot 
alcohol,  ether,  and  benzol  they  dissolve  with  ease.  Chemically 
speaking,  they  are  neutral  compound  ethers  which  are  formed 
through  the  union  of  an  acid  with  an  alcohol  according  to  the 
equation  : 

C2H5.OH  +  CH3.COOH  =  CH3.COO.C2H5  +  H20. 

The  fats  which  are  principally  found  in  the  animal  world,  viz., 
palmitin,  stearin,  and  olein,  similarly  result  through  the  union  of 
their  respective  monobasic  acids  with  the  triatomic  alcohol  glycerin. 
This  union  is  effected  as  shown  in  the  equations  : 

C3H5(OH)3  +  3C,5H31.COOH  =  C3H5(CI6H3102)3  +  3H20 
Glycerin.  Palmitic  acid.  Palmitin. 

C3H5(OH)3  +  3C17H35.COOH  =  C3H5(C18H3502)3  +  3H20 
Stearic  acid.  Stearin. 

C3H5(OH)3  +  3C17H33.COOH  =  C3H5(C18H3302)3  +  3H20 
Oleic  acid.  Olein. 

They  are  thus  triglycerides,  and  are  accordingly  termed  tripal- 
mitin,  tristearin,  and  triolein.  Other  fats  have  also  been  found  in 
the  animal  world,  but  are  of  secondary  importance.  Such  fats  are 
the  so-called  cetin,  which  is  obtained  from  certain  whales,  the  myricin 
of  beeswax,  etc. 

The  animal  fat  as  a  whole  usually  represents  a  mixture  of  the 
three  triglycerides,  palmitin,  stearin,  and  olein,  in  variable  propor- 
tions ;  the  stearin  predominating  in  the  more  solid  varieties,  while 
olein  prevails  in  the  more  liquid  fats.  In  human  fat  olein  represents 
about  670  to  800  pro  mille  of  the  total  amount. 

The  triglycerides  are  lighter  than  water ;  they  are  soluble  in 
benzol  and  ether,  and  in  hot  alcohol,  while  in  water  and  cold 
alcohol  they  are  insoluble.     They  are  non-volatile  and  burn  with  a 


THE  LECITHINS.  65 

luminous  flame.  On  heating,  especially  in  the  presence  of  potassium 
bisulphate,  they  are  decomposed  with  the  formation  of  highly  irritat- 
ing vapors  of  an  aldehyde,  acrolein,  which  in  turn  results  from 
glycerin,  according  to  the  equation  : 

C3H5(OH)3  =  C2H3.CHO  +  2H20. 

On  boiling  with  concentrated  alkalies,  or  through  the  influence 
of  superheated  steam,  as  also  through  certain  ferments,  the  fats  are 
decomposed  into  glycerin  and  their  respective  acids.  This  decom- 
position is  spoken  of  as  saponification,  and  the  alkaline  salts  of  the 
resulting  fatty  acids  are  accordingly  termed  "  soaps." 

On  prolonged  exposure  to  the  air,  even  in  the  absence  of  micro- 
organisms, the  tats  become  rancid — i.  e.,  they  become  acid  and  assume 
a  most  disagreeable  odor  and  taste.  During  this  process  a  partial 
decomposition  occurs,  with  the  formation  of  glycerin  and  fatty 
acids,  which  latter  are  then  oxidized  to  certain  volatile,  offensive 
smelling  oxy-acids.  The  exact  nature  of  the  process  which  thus 
takes  place  is  not  well  understood,  but,  as  has  been  stated,  it  "can 
occur  in  the  absence  of  micro-organisms,  and  through  the  influence 
of  light  and  air  only. 

The  fats  which  occur  in  the  animal  body  generally  present  a  more 
or  less  well-marked  yellow  or  red  color.  This  color  is  referable  to 
the  presence  of  certain  lipochromes.  These  are  compounds  which, 
like  the  fats  themselves,  are  devoid  of  nitrogen  ;  and  some  of  them 
apparently  are  hydrocarbons,  of  whose  structural  composition,  how- 
ever, nothing  is  known. 

Closely  related  to  the  fats  are  the  so-called  lecithins  and  choleste- 
rins.  The  latter  were  formerly  regarded  as  essential  food-stuffs  ; 
and  although  this  view  has  been  proved  erroneous,  they  are  never- 
theless considered  in  this  connection.  Some  of  the  lecithins,  on  the 
other  hand,  possess  a  distinct  nutritive  value. 

THE  LECITHINS. 

The  lecithins  are  ethereal  compounds  which  result  from  the  union 
of  cholin  with  glycerin-phosphoric  acid,  in  which  the  two  glycerin 
hydroxy]  groups  have  been  replaced  by  fatty  acid  radicles.  This 
union  takes  place  according  to  the  equations: 

1  I  -  II  .OH  CBLOH 

I  I 

<  II  "II        OH.PO.(OH),  =  CH.OH  +  11,0 

<  II  ..'HI  GRJO— PO(OH), 

Glycerin.  Glycerin-phosphoric  acid. 

(2;  (  II  .'»||  (  IMM.JI    " 

CH.OH       24     II   ,COOH      CH.O.CBHM0   |   2H,0 

GH*0— PO  "II  <  ll,.<>    PO(OH)j 

Di  Btearyl-glycerln-phoBphorlc 
acid. 


66  THE  FATS. 

(3)  CH2.O.C18H350  f  (CH3)3  CH2.O.C18H350 

I  II  I 

CH.O.Cl8H350  +  N  \    CH2— CH2.OH  =  CH.O.C18H350 

I  II  I 

CH2.0— PO(OH)2       L   OH  CH2.0— PO.C2H4 

Cholin.  OH    (CH3)3  J-  N 

OH 

Lecithin. 

On  decomposition  of  the  lecithins  with  acids  or  alkalies  we 
accordingly  obtain  glycerin-phosphoric  acid,  fatty  acids,  and  cholin. 
At  the  same  time,  however,  another  basic  substance,  ?ieurin,  is 
usually  found,  and  it  is  to  be  noted  that,  in  contradistinction  to 
cholin,  neurin  possesses  extremely  toxic  properties.  It  results  from 
cholin  through  the  loss  of  two  atoms  of  hydrogen  and  one  atom 
of  oxygen,  and  is  also  formed  during  bacterial  decomposition  of 
the    lecithins    in    the  presence  of  much  oxygen.     Chemically  it  is 

(CH3)3 
/ 
trimethyl-vinyl-ammonium  hydroxide,  N — CH=CH2,  while  cholin 

\ 
OH 

must  be  regarded  as  trimethyl-oxyethyl-ammonium  hydroxide. 

Another  derivative  of  cholin  which  may  be  obtained  through  the 
action  of  fuming  nitric  acid,  is  a  basic  substance  that  is  apparently 
isomeric  with  muscarin,  and,  like  this,  extremely  toxic.    Chemically 

(CH3)3 
/ 
it  may  possibly  be  represented  by  the  formula  N — CH2.COH,  and 

OH 

could  accordingly  be  regarded  as  the  aldehyde  of  oxyneurin  (tri- 
methyl  glycocoll). 

The  lecithin  which  is  most  commonly  found  in  the  aninial  body 
is  the  cholin  compound  of  distearyl-glycerin-phosphoric  acid  ;  other 
lecithins  can,  of  course,  also  occur,  in  which  the  glycerin  hydroxyl 
groups  have  been  replaced  by  the  radicles  of  oleic  and  palmitic  acids, 
for  example,  but  they  are  but  little  known. 

In  its  dry  state  the  common  lecithin  occurs  as  a  wax-like,  plastic 
mass,  which  is  soluble  in  alcohol  (at  40°-50°  C),  ether  (less  readily), 
chloroform,  benzol,  carbon  disulphide,  and  in  the  fatty  oils,  while  in 
water  it  is  insoluble.  Placed  in  water  it  swells  and  becomes  pasty, 
and  on  microscopical  examination  it  will  be  noted  that  the  substance 
occurs  in  the  form  of  peculiar  slimy  droplets  and  threads,  which  are 
generally  termed  its  myelin  forms.  From  its  alcoholic  solution  it 
crystallizes  in  wart-like  masses,  which  consist  of  small  platelets. 

Of  special  interest  is  the  tendency  of  the  lecithins  to  combine  with 
albumins  to  form  more  or  less  stable  compounds,  which  have  been 
termed  lecithalbumins.  Such  compounds  have  been  found  in  the 
mucosa  of  the  stomach,  in  the  lungs,  the  liver,  and  the  spleen.     In 


THE  CHOLESTERLXS.  67 

the  yolk  of  eggs  it  occurs  in  combination  with  vitellin,  but  is  here 
apparently  not  closely  bound.  A  certain  similarity  thus  exists 
between  the  lecithins  and  the  nucleins  ;  both  contain  phosphorus 
in  their  molecules,  and  both  combine  with  albumins  to  form  more 
complex  substances.  The  lecithins  occur  widely  distributed  in  both 
the  animal  and  vegetable  world.  According  to  Hoppe-Seyler,  they 
are  found  in  all  cells  and  bodily  fluids.  They  are  especially  abun- 
dant in  nerve-tissue  and  also  in  the  eggs  and  semen  of  most  animals. 
Their  isolation  and  special  tests  will  be  considered  in  a  subsequent 
chapter. 

THE  CHOLESTERINS. 

The  cholesterins  are  monatomic  alcohols  of  the  formula  C2-H45. 
OH  +  H20,  and  occur  widely  distributed  both  in  the  vegetable  and 
the  animal  world.  They  are  especially  abundant  in  nerve-tissue 
and  in  the  bile.  In  the  gall-bladder  they  are  frequently  found  in 
the  form  of  so-called  gall-stones,  and  not  uncommonly  constitute 
the  greater  portion  of  their  solids.  Different  varieties  apparently 
exist,  such  as  the  common  cholesterin  of  the  concretions  just  men- 
tioned, isocholesterin  (which  has  been  obtained  from  lanolin),  phy- 
tosterin,  paracholesterin,  and  kaulosterin,  which  are  found  in 
plants.  Their  structural  composition  is  unknown.  Like  the  fats, 
they  are  insoluble  in  water,  but  soluble  in  ether,  alcohol,  and  chloro- 
form, from  which  solutions  they  may  be  obtained  either  in  the  form 
of  very  characteristic  platelets  or  as  needle-like  crystals.  In  solu- 
tions of  the  alkalies,  in  the  absence  of  alcohol,  they  are  entirely 
insoluble,  even  on  boiling,  in  which  respect  they  differ  from  the  fats. 
Like  glycerin,  cholesterin  combines  with  fatty  acids  to  form  com- 
pound ethers,  and  in  this  form  it  is  frequently  found  in  nature.  In 
wool-fat,  for  example,  it  is  thus  present  in  large  amounts,  and  from 
it  such  ethers  can  be  readily  obtained.  In  pure  form  they  constitute 
the  lanolin  of  the  shops.  These  ethers  show  a  remarkable  difference, 
as  compared  with  the  fats,  in  their  behavior  toward  water.  Of  this 
they  apparently  take  up  one-quarter  of  their  own  weight,  and  on 
stirring  give  rise  to  a  pasty,  frothy  mass. 

From  their  ethereal  compounds  eholesterin  can  readily  be  sepa- 
rated by  treating  with  diacetic-ethy]  ether,  which  dissolves  the 
cholesterin  and  leaves  the  ethers  behind.  Their  special  tests,  and 
also  their  mode  of  isolation,  will  be  taken  up  in  a  subsequent 
chapter. 

After  having  thus   studied  the.  three  great  classes  of  food-stuffs 

which    plants  are  capable  of  elaborating  from  water,  carbon  dioxide, 

and  certain  mineral  -alt-,  and  which  are  also  represented  ill  the 
animal  body,  we  shall  now  proceed  to  a  similar  survey  of  the  natural 
decomposition-products  of  these  substances  which  are  formed  during 

their  passage  through  the  animal  body,  and  which  are  of  more  or  less 
interesl    in    indicating    the    manner    in    which    these   decompositions 


68  THE  FATS. 

are  effected.  Like  the  substances  that  have  already  been  con- 
sidered, these  products  will  be  taken  up  only  in  a  general  way  at 
first ;  while  their  special  study,  as  well  as  their  methods  of  isolation 
and  quantitative  estimation,  will  be  considered  in  succeeding  chapters, 
in  connection  with  the  chemical  study  of  those  tissues  in  which  they 
are  principally  encountered.  At  this  place  I  wish  only  to  impress 
general  facts  upon  the  mind  of  the  student,  so  that  he  will  be  pre- 
pared to  understand  the  composite  chemical  structure  of  the  various 
tissues  and  organs  of  the  body,  as  will  be  described  later. 


CHAPTER   V. 

THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 
THE   PROTAMINS. 

The  term  protamin  was  first  introduced  by  Miescher  to  designate 
a  basic  substance  which  he  was  able  to  isolate  from  the  spermatozoa 
of  the  salmon,  and  which  is  there  apparently  united  with  a  nuclemic 
acid  radicle.  Kossel  then  showed  that  a  very  similar  substance  can 
be  obtained  from  the  spermatozoa  of  the  sturgeon  ;  and  his  pupil, 
Kuraieff  isolated  a  third  body  of  this  order  from  the  spermatozoa 
of  the  mackerel.  It  was  thus  shown  that  different  protamins  occur 
in  nature,  and  the  use  of  the  term  has  since  been  extended  to  the 
entire  class.  Miescher's  original  protamin  is  now  spoken  of  as 
sXLand  is  identical  with  Kossel's  clupem,  which  was  obtained 
from  the  spermatozoa  of  the  herring.  The  two  other  protamms 
which  have  thus  far  been  isolated  are  termed  storm  and  scombnn, 
according  to  their  origin  from  the  sturgeon  and  the  mackerel,  re- 

SPA Jo^ding  to  Kossel,  the  protamins  are  essentially  albumins  of 
the  lowest  order,  and  he  assumes  that  a  radicle  of  this  kind  forms 
the  nucleus  of  all  the  more  complex  albumins.  This  assumption  is 
largely  based  upon  the  observation  that  all  protamins  yied  certain 
dewmposition-products,  which  may  also  be  obtained  from  the  various 


albumin 


aiounini:-.  ,  ,  •      i  • 

These  products  are  the  so-called  hexon  bases,  and  comprise  his- 
tidin  arginin,  and  lysin.  But  whereas  sturin  and  all  the  complex 
albumins  which  have  been  examined  in  this  direction  give  rise  to 
the  formation  of  all  three  of  these  bodies  apparently,  salmin  (clupem) 
and  scombrin  only  contain  the  arginin  group.  It  consequently  fol- 
lows that  the  protamin  radicle  of  the  complex'  albuminous  molecule 
mu.t  be  of  the  nature  of  sturin.  Whether  other  complex  albumins 
exisl  which  also  contain   the  salmin  or  Bcombnn   radicle  is  as  yet 

unknown.  ,  .      , 

\-  regards  the  quantitative  relations  which  exist  between  the  three 
hexon  bases  within  the  sturin  molecule,  our  knowledge  is  incom- 
plete. Kossel  suggested  that  six  molecules  here  unite  in  such-a 
manner  thai  every  two  combining  molecules  lose  two  molecules  ot 
water,  and  that  the  formation  of  sturin  might  bence  be  represented 
by  the  equation  : 

M!,no;      ::i,n„N40;  I  ^j!Mvo.      r:i6n«,N,A  |  5H20 

Hifltldin.  Arginin.  Sturin. 

However  this  may  he,  it   must  not  be  Hippo.., I  thai  the  quantita- 


.','.1 


70      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

tive  relations  between  the  hexon  bases  are  always  the  same,  and  as  a 
matter  of  fact  we  have  reason  to  assume  that  the  nucleus  of  most  of 
the  complex  albumins  contains  more  lysin  groups  than  are  found  in 
the  sturin  molecule. 

As  regards  the  further  development  of  the  complex  albumins 
from  protamins,  Kossel  imagines  that  these  are  formed  through  the 
union  of  a  protamin  radicle  with  various  other  radicles,  which  partly 
belong  to  the  class  of  the  mo  no-am  ido-acids,  and  partly  to  the  aro- 
matic series,  to  which  a  sulphur  or  an  iodine  radicle  can  further  be 
added.  These  various  bodies  can  then  also  combine  with  each  other 
or  with  foreign  radicles  to  form  still  more  complex  substances.  A 
relation  is  thus  established  between  the  lowest  forms  of  the  albumins 
and  the  carbohydrates,  and  just  as  the  polysaccharides  can  be  decom- 
posed by  hydrolysis  into  the  hexoses,  so  also  are  the  protamins  trans- 
formed into  hexons.  This  decomposition  in  both  instances,  more- 
over, can  be  effected  through  the  agency  of  ferments.  Diastase  thus 
causes  inversion  of  the  polysaccharides  to  hexoses,  and  trypsin  can 
similarly  break  down  the  protamins,  with  the  formation  of  the  hexon 
bases.  Whether  a  reversion  is  here  also  possible  has  not  as  yet  been 
ascertained. 

As  the  size  of  the  protamin  molecule  has  not  been  established,  it 
is  possible  only  to  indicate  the  composition  of  salmin,  sturin,  and 
scombrin  by  the  respective  formula?  (C30H57N17O6)a;,  (C36H69N1907)k, 
and  (C30H69N16O6)«. 

Like  the  complex  albumins,  the  protamins  give  the  bluish-violet 
biuret  reaction,  and  from  what  has  been  said  we  have  reason  to 
assume  that  this  reaction  in  the  case  of  all  albumins  is  referable  to 
the  presence  of  a  protamin  group.  The  xanthoproteic  reaction, 
Millon's  reaction,  Molisch's  reaction,  and  the  sulphur-test,  on  the 
other  hand,  are  negative. 

The  aqueous  solutions  of  the  protamins  are  strongly  alkaline  ; 
from  these  solutions  they  are  precipitated  by  picric  acid,  phospho- 
tungstic  acid,  iodopotassic  iodide,  potassium  ferrocyanide  in  the 
presence  of  acetic  acid,  by  salting  with  sodium  chloride  or  ammo- 
nium sulphate,  etc.  They  combine  with  acids  to  form  salts,  among 
which  the  sulphates  are  especially  characteristic,  as  on  cooling  or 
upon  the  addition  of  ether  they  separate  from  their  aqueous  solution 
in  the  form  of  an  oily  material.  With  the  salts  of  the  heavy  metals 
they  further  combine  to  form  double  salts.  With  the  coagulable 
albumins  and  the  so-called  primary  albumoses,  in  ammoniacal  solu- 
tion, the  protamins  combine  to  form  compounds  which  are  apparently 
identical  with  the  histons,  and  are  precipitated  as  such.  Neither  the 
protamins  nor  their  salts  have  thus  far  been  obtained  in  the  crystal- 
line state.1 

1  Quite  recently  Kurajeff  announced  that  he  has  been  able  to  isolate  pro- 
tamins also  from  the  mature  spermatozoa  of  the  pike,  of  Silurus  glanis  and  Acci- 
pensis  stallatus.  The  products  from  the  two  latter  he  has  termed  silurin  and 
accipenserin,  respectively.  For  the  accipenserin  sulphate  he  gives  the  formula 
C35H72N1809.4H2S04.     He  regards  it  as  closely  related  to  sturin. 


THE  HEX  ON  BASES. 


71 


Closely  related  to  the  protamins  on  the  one  hand,  and  to  the 
histons  on  the  other,  are  two  bodies  which  have  been  obtained  from 
the  spermatozoa  of  an  Arbacia  and  Cyclopterus  lumpus,  and  which 
are  termed  arbacin  and  cyclopterin,  respectively.  Of  these  cyclop- 
terin  is  more  closely  related  to  the  protamins  proper,  and,  like  these, 
yields  a  sulphate,  which  may  be  obtained  as  an  oily  material. 
Unlike  the  protamins,  however,  it  gives  Millon's  reaction,  but  does 
not  form  a  precipitate  on  heating;  on  cooling,  it  separates  out,  . 
and  then  presents  a  rose  color.  It  contains  much  less  oxygen  than 
the  protamins.  From  most  of  the  histons  it  differs  m  containing  no 
sulphur  and  in  not  being  precipitated  by  ammonia.  Its  formula  has 
not  as  vet  been  ascertained.  The  sulphate  contains  42.03  per  cent, 
of  carbon,  6.90  per  cent,  of  hydrogen,  and  22.08  per  cent,  of  nitrogen. 

Arbacin  differs  from  the  protamins  and  cyclopterin  in  containing 
much  less  nitrogen,  but,  like  cyclopterin,  it  gives  Millon's  reaction. 
It  is  not  completely  precipitated  from  its  solutions  by  ammonia,  but 
resembles  the  histons  in  other  respects.  Kossel  indeed  seems  now 
to  regard  it  as  such.  It  is  precipitated  from  its  solutions  bythe 
neutral  alkaloidal  reagents,  and  itself  precipitates  albumins.  It  gives 
the  biuret  reaction.1 

THE   PROTONS. 

The  protons  are  substances  closely  related  to  the  protamins,  and 
are  formed  as  intermediary  products  during  the  hydrolytic  decom- 
position of  the  latter  into  hexon  bases.  Individually  they  are 
but  little  known.  Thev  differ  from  the  protamins  in  the  greater 
solubility  of  their  sulphates  and  in  the  fact  that  they  are  not 
thrown  "down  bv  tin-  protamin  preeipitants,  or,  if  so,  are  more 
readily  soluble.  '  With  the  coagulable  albumins  and  the  primary 
albumoses  in  ammoniacal  solution,  moreover,  they  do  not  give  rise 
to  a  precipitate,  or  to  a  slight  turbidity  only,  which  may  be  due  to 
tracer  of  andecomposed  protamin.  From  clupein  three  protons 
have  been  obtained!  One  of  these  apparently  has  the  same  com- 
position aa  clupein  itself,  while  the  others  contain  more  hydrogen, 
but  less  carbon  and  nitrogen,  and  may  hence  be  regarded  as  clupein 
hydrates.  The  formula  of  one  of  these  is  ( ':;jHfi,\17<  >„  and  n  is 
interesting  bo  note  that  the  analogous  product  of  sturin  has  the  same 

composition. 

THE  HEXON  BASES. 

The  hexon   bases  comprise  arginin,  histidin,  and  lysin.     As  has 
been  stated,  they  are  formed  during  the  hydrolytic  decomposition  of 

i  From  Lota  vulgaris  Klirstrom  obtained  a  biBton-like  body,  which  be  terms  lota 

hwton    This  is  insoluble  in  water  and  solutions  of  the  neutral  sails,  but  dissolves  in 

acids  and  alkalies.    From  its  acid  solutions  il  is  precipitated  by  ammonia.    It  gives 

armlet  biuret  reaction.   The  xanthoproteic  reaction  a  positive,  that  ol   Millui.  h-.-M. 

but  distinct    Molisch's  reaction  is  quite  intense,  and  thai  of  Adamkiewicz  uigni. 

\   -i„,ihr  body  was  found  by  kossel  in  the  mature  testicles  of  Gadus  marrhuft, 

I  by  Bang  in  the  immature  organs  of  the  mackerel.    The  twoarespoken  of 

biston  and  »combron,  respectively. 


72      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

the  protamins,  as  also  of  all  complex  albumins,  both  of  animal  and 
vegetable  character,  which  have  been  examined  in  this  direction. 
Among  these  may  be  mentioned  fibrin,  keratin,  spongin,  collagen, 
conglutin,  elastin,  egg-albumin,  casein,  etc.  Kutscher,  moreover, 
obtained  these  bodies  among  the  final  decomposition-products  of 
tryptic  digestion,  and  showed  that  that  portion  of  Kiihne's  anti- 
peptone  which  can  be  precipitated  with  phosphotungstic  acid  con- 
sists to  the  extent  of  from  30  to  31  per  cent,  of  these  hexon  bases. 

The  free  bases,  like  the  protamins,  are  lsevorotatory,  while  their 
salts,  which  are  formed  through  union  with  acids  and  salts  of  the 
heavy  metals,  are  dextrorotatory.  These  salts  can  be  obtained  in 
crystalline  form,  and  it  has  thus  been  possible  to  determine  the 
formulae  of  the  free  bases. 

Arginin. — On  hydrolytic  decomposition  arginin  yields  urea  and 
Ornithin.  The  substance  is  therefore  regarded  as  a  guanidin  deriva- 
tive, similar  to  kreatin,  in  which  one  amido-group  has  been  replaced 
by  the  ornithin  radicle.  The  correctness  of  this  supposition  has 
since  been  established  through  the  synthesis  of  arginin  from  cyana- 
mide  and  ornithin.  Ornithin,  moreover,  is  now  known  to  be  a, 
o-diamido-valerianic  acid,  and  the  structural  formula  of  arginin 
may  hence  be  represented  as  follows  : 

NH2  NH, 

I  I 

NH  =  C  —  NH.CH2.CH2.CH2.CHCOOH 

Its  relation  to  guanidin  and  kreatin,  as  also  its  decomposition  into 
urea  and  ornithin,  is  further  shown  : 

/NH2  .NH, 

Cf(NH)  <(NH) 

\nH,  \N(CH3).CH2.COOH 

Guanidin.  Kreatin. 

/NH, 
CN.NH,  +  CH(CH,).NH2.COOH  =  C^(NH) 
Cyanamide.  Methy'l-glycocoll.  \N(CH3).CH2.COOH 

Kreatin. 

/NH, 
CN.NH,  +  CH2.CH2.CH2.CH.COOH  =  C^(NH) 

|  |  \NH.CH2.CH2.CH2.CH.COOH 

Cyanamide.     NH2  NH2  I 

Ornithin.   "  NH2 

Arginin. 
NH2 
Cf(NH) 
\NH.CH2.CH2.CH,.CH(NH2).COOH  +  HO  = 

Arginin. 

NH2 
CO(  +  CH2(NH2).CH2.CH2.CH(NH2)COOH 

^NH,  Ornithin. 

Urea. 

On  oxidizing  arginin  with  barium  permanganate  Kutscher 
obtained  guanidin,  guaniclin-butyric  acid,  and  ethylene-succinic  acid. 
He  concludes  that  the  process  occurs  in  two  phases,  as 'represented 
by  the  equations  : 


THE  HEXON  BASES.  73 

/NH,  NH2 

(1)  (T.NH  |  +20  = 

\NH.  CH2.CH2.  CH,.  CH.  COOH 
Arginin. 

/NH2 

C(-  Nil  +  C02  +  NH3 

\NH.  CH,.CH2.CH2.COOH 

Guanidin-butyric  acid. 

/NH2 

(2)  (T.NH  +  20  = 

NII.CH2.CH2.CH2.C(X)H 
Guanidin-butyric  "acid. 

/NE, 
C(-NH  +  C00HCH2.CH2  COOH 
\\H,  Succinic  acid. 

Guanidin. 

Of  great  interest,  further,  is  the  fact  that  ornithin  can  give  rise 
to  putrescin,  viz.,  to  tetramethylene-diamine,  a  ptomain  which  is 
formed  during  the  putrefaction  of  albuminous  material,  and  which 
has  also  been  found  in  the  urine  in  association  with  cyst  in.  Thus 
far  this  transformation  has  been  effected  only  through  the  agency  of 
micro-organisms,  but  there  is  no  reason  to  suppose  that  their  presence 
is  essential,  and  that  in  the  tissues  of  the  living  body  the  same  proc- 
ess cannot  also  occur.  This  transformation  may  be  represented  by 
the  equation  : 

CH2(NH2).CH,CH,,CH.(NH2).COOH  =  C02  +  CH2(NH,).CH.,CH2.CH2.(NH2) 
Ornithin.  Putrescin. 

Should  the  formula  of  ornithin,  as  above  indicated,  be  correct — 
and  it  may  be  added  that  there  is  every  reason  to  suppose  that  this 
is  the  case — we  can  readily  understand  how  pyridin  derivatives  can 
develop  from  the  albumins  without  being  forced  to  assume  the 
existence  of  a  pyridin  radicle  in  the  albuminous  molecule  directly. 
The  active  principle  of  the  suprarenal  gland,  which  von  Fiirth 
regards  as  tetrahydro-dioxypyridin,  could  thus  result  from  ornithin 
by  tli«'  replacement  of  the  a-amido-group  by  hydroxy]  and  the  elim- 
ination of  water.  That  oxypiperidin  results  from  <5-amido-valerianic 
acid  in  an  analogous  manner  lias  indeed  been  demonstrated.  These 
relations  may  he  expressed  by  the  formulae: 

•II     Ml    .<  I[,.(  ll,cil,.(0()ll        ||(»       ( 'H,i  Ml  i.UU'l  !.,.<']  I2.CO. 
8-amido-valerianic  acid.  oxypiperidin. 

I)   CH    Ml     «  II  .1  Il..<  ll.MU.COOH         II,o 

Ornithin.  <   1 1.,(  M  I  .  ..<   I  I     <    I  I    <    I  I   Ol  I  i.<  <  ><>I  I     |     Ml,. 

a-hydroxy,  i  amido  valerianic  acid. 

2    'II    Ml,  .'  HPCHa.CH(OH).OOOH 

II.o   ■   <ii,-Mi,rii,<||,.cil  niii.n. 
i  el  rahydro-dioxypyridin. 

Lysin. — Lysin  is  apparently  :i  homologue  of  ornithin,  and  is 
represented  by  the  formula  CH2(NH2).CH2.CH2.CH2.CH(NH2). 
OOOH;    it     U    thus   '/,  e-diamido-capronic    acid.     <)n    hydrolytic 


74      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

decomposition  it  yields  ammonia,  oxalic  acid,  propionic  acid,  and 
notably  acetic  acid.  When  exposed  to  the  influence  of  putrefac- 
tive organisms  it  gives  rise  to  the  formation  of  cadaverin — penta- 
methylene-diamin — a  ptomain  which  is  frequently  found  together 
with  putrescin  in  putrefying  albuminous  material,  and,  like  this,  may 
also  appear  in  the  urine  in  association  with  cystin.  Its  formation  is 
quite  analogous  to  that  of  putrescin  from  ornithin,  and  may  be 
represented  by  the  equation  : 

CH2(NH2).CH2.CH2.CH2.CH(NH2).COOH  = 

Lysm.  C02  +  CH2(NH2).CH2.CH2.CH2.CH2(NH2) 

Cadaverin. 

In  this  manner,  also,  albumins  can  give  rise  to  the  formation  of 
pyridins,  and,  as  a  matter  of  fact,  piperidin  results  during  the  dry 
distillation  of  cadaverin,  as  represented  by  the  equation  : 

CH2 — CH2 

/  \ 

CH2(NH2).CH2.CH2.CH2.CH2(NH2)  =  CH2  NH  +  NH3. 

\  / 

CH2 — CH2 

On  treating  lysin  with  benzoyl  chloride  Drechsel  obtained  a 
body,  of  the  formula  C6H12(COC6H5)2N202,  which  he  termed  lysurio 
acid,  and  which  is  thus  homologous  with  the  dibeuzoyl  derivative 
of  ornithin,  C5H10(COC6H5)2N2O2,  ornithurie  acid. 

Histidin. — Of  the  nature  of  histidin  comparatively  little  is 
known.  This  is  largely  owing  to  the  fact  that  the  substance  is 
formed  only  in  very  small  amounts  during  the  decomposition  of 
albumins.  From  200  grammes  of  antipeptone  Kutscher  thus  ob- 
tained only  1.4  grammes  of  histidin,  as  compared  with  10.4  grammes 
of  arginin.     Its  formula,  according  to  Kossel,  is  C6H9N302. 

THE  NUCLEINIO  ACIDS. 

In  a  preceding  chapter  it  was  pointed  out  that  the  so-called 
nucleins  can  be  divided  into  two  classes,  viz.,  into  the  paranu- 
cleins,  or  pseudonucleins,  and  into  the  true  nuclear  nucleins.  It 
was  shown,  moreover,  that  in  the  nuclear  nucleins  an  albuminous 
radicle  is  combined  with  organic  phosphorus-containing  acids,  the 
so-called  nucleinic  acids.  Our  knowledge  of  these  bodies  is  very 
limited,  and  a  satisfactory  classification  impossible.  For  con- 
venience' sake  I  divide  the  nucleinic  acids  into  two  groups,  viz., 
the  primary  acids,  which  occur  in  nature  either  free  or  in  com- 
bination with  albumins  (including  the  protamins),  and  the  sec- 
ondary acids,  which  result  from  decomposition  of  the  primary  acids. 
These  latter  are  characterized  by  the  fact  that  on  decomposition 
they  all  yield  nucleinic  bases,  while  this  is  not  necessarily  the  case 
as  regards  the  secondary  acids.  According  to  their  origin,  these 
primary  acids  have  been  termed  spermanucleinic  acid,  thymo- 
ma cleinic  acid,  yeast-nucleinic  acid,  etc.     There  is  reason  to  assume, 


THE  NUCLEINIC  ACIDS.  75 

however,  that  these  acids  actually  represent  mixtures  of  different 
nucleinic  acids,  and  Kossel  expresses  the  opinion  that  in  reality 
onlv  four  true  nucleinic  acids  exist,  viz.,  adenylic  acid,  guanylic 
acid,  sarcylic  (hypoxanthylic)  acid,  and  xanthylic  acid.  He  further 
believes  that  only  one  nucleinic  base  is  represented  in  each  one  of 
these  acids,  viz.,  adenin,  guanin,  hvpoxanthin,  and  xanthin.  In 
accordance  with  this  supposition,  the  spermanucleinic  acid  of  the 
ox  would  contain  three  acids,  as  on  decomposition  it  yields  xan- 
thin, hvpoxanthin,  and  adenin.  Thymonucleinic  acid,  from  which 
only  adenin  and  g.uanin  have  been  isolated,  would  similarly  repre- 
sent a  mixture  of  adenylic  acid  and  guanylic  acid,  etc.  This 
assumption  of  Kossel,  however,  has  not  proved  correct,  for  we 
now  know  that  his  adenylic  acid,  for  example,  contains  not  only 
adenin,  but  also  guanin  and  a  third  basic  substance  which  has  been 
termed  cytoshi  Bang,  on  the  other  hand,  has  shown  that  a  nucleinic 
acid  can  be  isolated  from  the  pancreas  which  contains  only  one 
nucleinic  base,  guanin,  and  which  would  thus  correspond  to  Kossel's 
hypothetical  guanylic  acid.  Then,  again,  it  appears  that  the  so-called 
inosinifi  aeid,  which  has  been  found  in  muscle-tissue,  contains  only 
hvpoxanthin.  But  we  see  nevertheless  that  more  than  one  of  the 
nucleinic  bases  may  occur  in  the  molecule  of  one  nucleinic  acid. 

All  nucleinic  acids  contain  carbon,  hydrogen,  nitrogen,  oxygen, 
and  a  large  percentage  of  phosphorus,  of  which  indeed  one  part  is 
usually  found  for  every  three  parts  of  nitrogen.  Sulphur  is  not 
present.  Of  the  form  in  which  the  phosphorus  exists  in  the  nucle- 
inic acid  molecule  but  little  is  known.  The  assumption  that  the 
true  nucleins  represent  compounds  of  albumins  with  metaphos- 
phoric  acid,  to  which  metaphosphates  of  xanthin  and  guanin  are 
admixed,  is  no  longer  tenable.  According  to  Kossel,  the  nucleinic 
acids  possess  a  radicle  which  contains  a  number  of  phosphorus 
atom-  united  to  each  other  after  the  manner  of  the  polymetaphos- 
phoric  acids,  and  the  evidence  is  now  conclusive  that  the  nucleinic 
bases  are  present  in  the  nucleinic  acid  radicles  as  organic  com- 
pounds. Of  special  interest,  further,  is  the  fact  that  some  of  the 
nucleinic  acids  contain  a  carbohydrate  group.  From  yeast-nucleinic 
acid  Kossel  was  thus  able  to  obtain  a  hexose  as  well  as  a  pentose. 
In  guanylic  acid  a  pentose  is  also  apparently  present,  and  from  the 
spermanucleinic  aeid  of  the  sturgeon,  as  also  from  thymonucleinic 
acid,  laevulinic  aeid  can  be  obtained. 

A-  regards  the  structural  composition  of  the  individual  nucleinic 
acids,  our  knowledge  is  very  incomplete.  The  general  formulae  of 
tin-  more  importanl  members  of  the  group  are  here  given: 

Spermanucleinic  acid  of  the  salmon ('io"m^ti4(  Nt-''  '  N 

Yeas!  nucleinic  acid <  ",0 1  IS!) N  ,,;<  >.,./_' I ' ..<>, 

Thymonucleinic  acid '  ,}  l:n;\,<  >.,„l's 

Guanylic  acid <  •..■.."31^ 'm(  W^ 

[nosinic acid      <  'i„l  l| ,;N ' ,' > .  I' 

On   decomposition   the  primary   nucleinic  acids  give  rise  to  the 


76      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

formation  of  what  I  have  termed  the  secondary  nucleinic  acids. 
These  contain  more  phosphorus  than  the  primary  acids,  and  may  or 
may  not  give  rise  to  xanthin  bases  on  further  decomposition.  They 
may  accordingly  be  divided  into  acids  of  the  type  of  plasminic  acid 
and  of  thyminic  acid,  respectively.  The  former  result  from  the 
primary  acids  through  a  splitting  off  of  atomic  groups,  which  are 
free  from  phosphorus,  but  ultimately  the  characteristic  decomposi- 
tion-products of  the  primary  acids  result,  viz.,  the  nucleinic  bases, 
phosphoric  acid,  etc. 

Thyminic  acid,  on  the  other  hand,  is  obtained  from  the  primary 
acids,  with  the  possible  exception  of  inosinic  acid  and  guanylic 
acid,  after  the  separation  of  the  nucleinic  bases.  For  its  barium 
salt  Kossel  obtained  the  formula  C16H23N3P2012Ba.  On  decomposi- 
tion with  strong  sulphuric  acid  thyminic  acid  yields  a  crystalline 
product  termed  thymin.  As  this  substance  is  obtained  as  a  con- 
stant decomposition-product  from  all  the  primary  nucleinic  acids 
which  have  been  examined  in  this  direction,  with  the  exception, 
as  has  been  indicated,  of  inosinic  acid  and  guanylic  acid,  Kossel 
now  divides  these  primary  acids  into  thymonucleinic  acids,  and 
a  second  group  which  is  represented  by  the  two  exceptions  just 
mentioned. 

Thymin  (C5H6]N!"202),  according  to  Steudel,  may  be  regarded  as  a 
methyl-dioxy-pyrimidin,  and  is  thus  isomeric  with  methyl-uracil. 
It  is  closely  related  to  the  ureids  (barbituric  acid,  etc.)  and  the  purin 
bases  (see  below),  and  it  is  possible,  as  a  matter  of  fact,  to  condense 
isodialuric  acid,  which  is  closely  related  to  uracil,  with  urea  to  uric 
acid.  The  position  of  the  hydrogen  and  oxygen  atoms,  as  also  of 
the  methyl  group,  in  their  relation  to  the  pyrimidin  nucleus,  is,  how- 
ever, as  yet  uncertain.  The  relation  existing  between  these  bodies 
may  be  seen  from  the  following  formulae : 

N  NH  N 

/-\  /\  /\ 

CH        CH  CO        C— CH3  CH        CH 

I           II  I           II                              II 

N        CH  NH        CH                            N        C— NHX 

\/  \/                                    W             >CH 

CH  CO                                        C N^      - 

Pyrimidin.  Methyl-uracil.                             .          Purin. 

The  primary  nucleinic  acids  are  amorphous  and  possess  a  strongly 
acid  reaction.  They  are  soluble  in  dilute  solutions  of  the  alkalies, 
and  are  precipitated  from  these  solutions,  especially  in  the  presence 
of  alcohol,  by  adding  a  slight  excess  of  hydrochloric  acid.  In 
alcohol  and  ether  they  are  insoluble.  In  acid  solutions  (acetic;  acid) 
they  give  rise  to  precipitates  with  albumins,  which  are  apparently 
identical  with  the  nucleins.  From  the  nucleins  they  are  obtained 
by  treating  with  a  dilute  solution  of  sodium  hydrate,  which  is  subse- 
quently neutralized  with  dilute  hydrochloric  acid.  The  separated 
albumin  is  then  precipitated  by  adding  an  excess  of  acetic  acid,  when 


THE  NVCLEINIO  BASES.  77 

the  filtrate  is  treated  with  an  equal  volume  of  alcohol  and  hydro- 
chloric acid  to  the  extent  of  from  3  to  5  pro  mille.  In  this  manner 
impure  nucleinic  acid  is  thrown  down,  which  can  be  further  purified 
by  solution  in  ammoniacal  water  and  further  treatment  with  acetic 
acid,  hydrochloric  acid,  and  alcohol,  as  just  described. 

Thyminic  acid  differs  from  the  nucleinic  acids  proper  in  its  ready 
solubility  in  cold  water,  and  in  the  fact  that  it  is  not  precipitated 
from  its  solutions  by  the  mineral  acids.  Like  the  nucleinic  acids,  it 
gives  a  precipitate  with  albumins  or  primary  albumoses  (propep- 
tones)  in  acetic  acid  solution,  but,  in  contradistinction  to  the  nucleinic 
acids,  this  precipitate  is  soluble  in  hydrochloric  acid  and  in  solu- 
tions of  many  salts. 

Plasminic  acid  likewise  precipitates  albumins  in  acid  solution, 
but,  unlike  the  nucleinic  acids, is  easily  soluble  in  water;  on  treating 
with  ammonia  a  yellow  color  develops.  Its  phosphoric  acid  radicle 
is  capable  of  binding  iron  in  such  form  that  it  appears  like  a  true 
organic  iron  compound.  According  to  Ascoli,  the  substance  contains 
1  per  cent,  of  iron.  It  does  not  give  Millon's  reaction  nor  the 
biuret  reaction,  and  contains  no  sulphur.  On  decomposition  with 
mineral  acids  by  boiling  it  yields  nucleinic  bases  and  phosphoric 
acid.     The  substance  may  be  obtained  from  yeast. 

The  question  whether  the paranucleim  contain  an  acid  radicle  which 
is  analogous  to  the  nucleinic  acids  is  still  undecided.  If  they  occur, 
such  paranucleinic  acids,  as  they  would  be  termed,  could,  of  course, 
not  contain  any  basic  radicle  of  the  character  of  the  nucleinic  bases. 
A  few  isolated  observations  seem  to  show  that  such  acids  exist. 
Altaian  thus  obtained  an  acid  from  the  yolk  of  eggs  which  he  re- 
garded as  a  nucleinic  acid.  As  the  nucleinic  bases,  however,  cannot 
be  obtained  from  the  same  source,  it  follows  that  the  substance  in 
question  could  not  be  a  true  nucleinic  acid.  Wildenow  further 
speaks  of  a  phosphorus-containing  substance,  which  she  was  able  to 
obtain  from  the  paranuclein  of  casein,  and  which  precipitated 
albumins.  Neither  of  these  bodies,  however,  has  as  yet  been 
isolated  in  a  form  suitable  for  analysis.  Whether  KossePs  SO- 
called  paranucleinic  acid,  which  was  later  shown  to  be  the  same  as 
thymonucleinic  acid,  is  identical  with  the  prosthetic  group  of  the 
paranuclein-,  remains  to  be  seen.  Its  properties  certainly  are  such 
as  would  a  •priori  be  expected  from  a  true  paranucleinic  acid.  But 
even  if  a  group  of  this  order  were  found  in  some  of  the  paranucleus, 
it-  presence  in  all  would  not  necessarily  follow,  and  it  is  quite  con- 
ceivable  that  in  others  the  albumin  is  directly  combined  with  a  phos- 
phoric  acid. 

THE  NUCLEINIC  BASES. 

The  nucleinic  bases,  which  are  also  spoken  of  as  the  xanthin, 
aUoxuriCf  or  purin  bases,  are  found  widely  distributed  both  in  fix; 
animal  and  the  vegetable  world.  They  occur  either  in  the  lice 
state   or   a-    OODStitUtente  of   tin;    nucleinic   acids   and    the    nucleins. 


78      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

They  comprise  xanthin,  hypoxanthin  (or  sarcin),  episarcin,  hetero- 
xanthin, paraxanthin,  theophyllin,  theobromin,  caffein,  guanin,  epi- 
guanin,  adenin,  and  carnin.  Of  these,  paraxanthin,  heteroxanthin, 
epiguanin,  and  episarcin  have  thus  far  been  found  only  in  the  urine. 
Theophyllin,  theobromin,  and  caffein  are  confined  to  the  vegetable 
world,  while  the  remaining  members  of  the  group  are  common  con- 
stituents of  both  animal  and  vegetable  cells.  All  these  bodies  are 
closely  related  to  each  other  and  to  uric  acid,  which,  as  we  shall  see 
later,  constitutes  one  of  the  most  important  end-products  of  animal 
metabolism.  According  to  Emil  Fischer,  they  are  derived  from  a 
hypothetical  substance,  which  has  been  termed  purin,  and  it  has 
previously  been  pointed  out  that  in  this  manner  a  further  relation- 
ship is  established  between  the  nucleinic  bases  and  thymin,  as  it  is 
probable  that  this  substance,  or  allied  bodies,  constitute  the  ante- 
cedents of  the  purin  radicle.  Behrend,  in  fact,  has  pointed  out  that 
through  the  condensation  of  urea  and  isodialuric  acid,  which  is 
closely  related  to  uracil,  uric  acid  results.  These  relations  are  shown 
below : 

(6) 

(l)  N^=CH 

I  I          (V) 

(2)HC      (5)C NHs 

||  >CH(8)  Purin  =  C5H4N4. 

(3)  n C N  * 

(4)  0) 

By  substituting  the  group  NH2  for  the  hydrogen  atom  at  6,  adenin 
results,  and  is  hence  spoken  of  as  a  6-amino-purin  : 

N C.NH2 

I  I 

HC         C NHX 

I!  ||  >CH  Adenin  =  C5H5N5. 

N C N  *     ■ 

Hypoxanthin,  according  to  this  conception,  would  be  _  6-oxypurin, 
xanthin  2,  6-dioxypurin,  and  guanin  2-amino-6-oxypurin : 

HN CO  HN CO 

II  II 

HC         C NJEL  CO         C NH. 

II  I  >CH  |  ||  >CH 

N C N  "  HN C N  V 

Hypoxanthin  =  C5H4N4O.  Xanthin  =  C5H4N4O2. 

HN CO 

I             I 
HN=C  C NHX 

I  II  >H 

HN C N  * 

Guanin  =  C6H5N60. 

From  these  primary  bodies  the  remaining  ones  then  result  through 
a  substitution  of  methyl  groups.  Heteroxanthin  is  thus  formed 
from  xanthin  by  replacing  the  hydrogen  atom  at  7  with  CH3,  and 
is  therefore   7-methyl-2,6-dioxypurin.     Paraxanthin  is  accordingly 


THE  NUCLEINIO  BASES.  79 

l,-7-dimethyl-2,6-dioxypurin,  and  caffein  l,3,7-trimethyl-2,6-dioxy- 
purin.  Theophyllin  and  theobromin  are  isomeric  with  paraxanthin, 
and  structurally  l,3-dimethyl-2,6-dioxypurin  and  3,7-dimethyl-2,6- 
dioxypurin,  respectively.  Epiguanin  is  similarly  derived  from 
guanin  by  the  replacement  of  the  hydrogen  atom  at  7  with  methyl, 
and  is  hence  7-methyl-guanin. 

HX CO 

CO         C N.CH3X 

HX C X^^ 

Hetcroxanthin  =  C6H6N4Oo. 


Caffein 

CH3.X CO 

Co        C XHX  CO        C X.CH3 

CH,.X C X    *  CH3.X C N^^ 

Theophyllin  =  C7H8N402.  Theobromin  =  C7H8N402. 

HX CO 

HX  =  C  C X.CH3X 

I            II  ^CH 

HX C X^^ 

Epiguanin  =  C6H7N50. 

Carnin  is  apparently  closely  related  to  hypoxanthin,  as  on  treatment 
with  bromine  it  yields  methyl  bromide,  carbon  dioxide,  and  the  brom- 
hydrate  of  hypoxanthin,  according  to  the  equation  : 

C7HsX4Os  +  2Br  =  C5H4X4O.HRr  +  CH3Br  +  C02. 

Its  structural  formula  is  thus  possibly  the  following : 

(II  .X CO 

I  I 

CHOH  ( XH  v 

I  XCH 

1 1 X-CO  =  CO X  ^ 

Episarcin  is  but  little  known.  Its  formula  is  given  as  C4H6NsO, 
but  it  is  possible  that  this  is  not  correct.  The  substance  has  un- 
doubtedly many  properties  in  common  with  epiguanin,  and  future 
researches  in<b<< j  may  possibly  show  that  the  two  are  identical. 

These  various  nueleinic  bases,  which  Gautier  also  designates  as 
the  xanthin  leucomaims,  have  the  character  of  feeble  alkaloids,  and 
Combine  with  hydrochloric  acid  and  platinum  chloride  to  form 
crystalline  -alt-,  which  are  dissociated  only  very  slowly  or  not  at  all 
by  water.  When  fused  with  alkalies  they  lose  the  greater  portion 
of  their  nitrogen  in  the  form  of  cyanogen,  and,  as  a  matter  of  fact, 


80      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

we  find  that  all  of  them  contain  the  group  HCN.  Adenin,  indeed, 
is  a  polymeric  compound  of  hydrocyanic  acid,  and  xanthin  can  be 
obtained  by  direct  hydration  of  the  same  body  (Gautier).  On  heat- 
ing with  alkalies  or  with  water  the  nucleinic  bases  generally  do  not 
give  rise  to  the  formation  of  urea,  and  they  cannot  hence  be  re- 
garded as  ureids,  although  a  close  relationship  exists  between  them. 
By  a  simultaneous  process  of  oxidation  and  hydration  guanin  thus 
gives  rise  to  parabanic  acid,  and  it  is  possible  indeed  to  pass  from 
guanin  to  xanthin  and  hypoxanthin  by  a  series  of  simple  reactions 
without  the  intervention  of  urea.  These  changes  are  represented  by 
the  equations  : 

/NH2       CO— NHX 
C5H5N50  +  30  +  H20  =  C(NH)<  +    |  >CO  +  C02 

Guanin.  \NH2         CO— NEK 

Guanidin.  Parabanic  acid. 

C5H5N50  +  HN02  =  C3H4N402  +  2N  +  H20. 
Guanin.  Xanthin. 

C5H4N402  +  2H  =  C5H4N40  +  H20. 
Xanthin.  Hypoxanthin. 

Xanthin,  however,  on  oxidation  and  hydration  yields  urea  and 
alloxan,  which  latter  is  further  oxidized  to  parabanic  acid  and 
carbon  dioxide,  as  shown  in  the  equations : 

CO-NHx 

I  \  /NH, 

C5H4N402  i-  20  +  H20  =  CO  >CO  +  CO< 

Xanthin.  |  /  XNH2 

CO— NH/  Urea. 

Alloxan. 

CO— NHX 

|  \  CO— NH, 

CO  >CO  +  0=|  >co  +  co2 

I  /  CO— nh/ 

CO— NET 

Alloxan.  Parabanic  acid. 

The  close  relationship  which  exists  between  uric  acid  and  the  nucle- 
inic bases  thus  becomes  apparent,  as  uric  acid  on  oxidation  and 
hydration  gives  rise  to  the  same  products  as  xanthin : 

C5H4N403  +  O  +  H20  =  C4H2N204  +  CON2H4. 
Uric  acid.  Alloxan.  Urea. 

This  relationship  is  further  shown  by  decomposing  the  primary 
xanthin  bases  and  uric  acid  Avith  fuming  hydrochloric  acid  or 
hydriodic  acid  under  high  pressure.  Qualitatively  the  same  prod- 
ucts are  thus  obtained,  viz.,  ammonia,  carbon  dioxide,  glycocoll, 
and  formic  acid,  while  the  quantitative  relations,  of  course,  vary 
with  the  nature  of  the  individual  substance. 

C5H5N5      +  8H20  =  4NH3  +    C02  4-  CH2.NH2.COOH  +  2H.C00H. 
Adenin.  Glycocoll.  Formic  acid. 

C5H4N40    -I-  7H20  =  3NH3  +    C02  +  CH2.NH2.COOH  +  2H.C00H. 
Hypoxanthin. 


THE   UREIDS.  81 

C5H5X50   +  7H20  =  4NH,  +  2C02  +  CH2.NH2.COOH  +  H.COOH. 

Guanin. 

C;H(XA  -  6H20  =  3NH3  +  2C02  +  CH2.NH2.COOH  +  H.COOH. 

Xanthin. 
( ,H4X  A  +  5H20  =  3NH,  +  3C02  +  CH2.NH2.COOH. 

Uric  acid. 

According  to  Emil  Fischer,  the  structural  formula  of  uric  acid  can 
be  represented  as  follows  : 

HX  —  CO 

CO    C— NHX 

I      I        >co 

HN—  C— XHX 

Uric  acid. 

and  it  is  thus  seen  that,  like  the  nucleinic  bases,  it  contains  the 
purin  radicle. 

All  the  nucleinic  bases  combine  with  acids  and  alkalies  to  torm 
salts,  many  of  which  are  readily  crystallizable.  On  boiling  with 
acetate  of  copper  most  of  them  are  thrown  down  as  insoluble  com- 
pounds. From  their  neutral  solutions,  or  in  the  presence  of  a  little 
ammonia,  they  are  precipitated  by  ammoniacal  silver  nitrate  solu- 
tion. Tin's  precipitate  dissolves  in  nitric  acid  on  the  application  of 
heat,  but  reappears  on  cooling,  Most  of  the  nucleinic  bases,  more- 
over, when  evaporated  to  dryness  in  the  presence  of  nitric  acid, 
leave  a  yellowish  residue,  which  changes  to  orange  and  often  to  a 
temporary  purple  on  the  addition  of  an  alkali.  In  this  respect  also 
these  substances  resemble  uric  acid,  which  gives  a  very  similar  reac- 
tion (see  Murexid  test).  When  exposed  to  the  action  of  putrefactive 
organisms  adenin  is  transformed  into  hypoxanthin,  and  guanin  into 
xanthin,  so  that  these  two  only  are  found  in  decomposed  material. 

For  a  description  of  the  individual  members  of  this  group  which 
are  found  in  the  animal  body,  as  well  for  the  method  of  their  isola- 
tion and  quantitative  estimation,  the  reader  is  referred  to  subsequent 
chapters  (see  especially  pages  241  and  363). 

THE  UREIDS. 

The  ureids  comprise  a  number  of  nitrogenous  crystallizable  bodies 
characterized  by  the  fact  that  on  hydrolytie  decomposition  alone,  or  on 
simultaneous  oxidation,  they  yield"  urea.  They  are  hence  derivatives 
of  urea,  and  may  contain  one  or  more  molecules  of  this^ substance,  in 
which  one  ormore  hydrogen  atom-  are  replaced  by  radicles  of  mono- 
basicor  polybasic  acid-.  On  decomposition  they  yield  either  urea 
and  a  oon-nitrogenous  acid  directly,  or  they  give  rise  to  urea  and  a 
less  complex  iireid,  which  is  then  further  decomposed,  as  in  the 
li,M  instance.  They  are  accordingly  divided  into  mono-ureids  and 
di-ureids.  The  former  generall)  contain  two  atoms  of  nitrogen  in 
their  molecule,  while  the  latter  possess  four  atoms  of  nitrogen.  All 
,!„._,.  bodies  are  closely  related  to  each  other  and  to  the  nuclemio 
bases,  from  which  they  are,  in  part  at  least,  derived. 


82      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

The  mono-ureids  comprise  alloxan,  alloxanic  acid,  dialuric  acid, 
barbituric  acid  or  rnalonyl-iirea  and  its  derivatives,  all  of  which  can 
be  decomposed  into  urea  and  non-nitrogenous  acids  containing  three 
atoms  of  carbon.  From  these  another  series  of  mono-ureids  is  de- 
rived, which  also  yield  urea  and  non-nitrogenous  acids  ;  but  the 
latter,  in  contradistinction  to  the  first  group,  contain  only  two  atoms 
of  carbon.  These  include  parabanic  acid,  oxaluric  acid,  allanturic 
acid,  and  hydantoin  and  its  derivatives,  viz.,  hydantoic  acid  or  glyco- 
luric  acid,  and  methyl-hydantoin.  The  di-ureids  are  similarly 
divided  into  two  groups,  the  first  of  which  comprises  uric  acid, 
alloxantin,  murexid  or  ammonium  purp urate,  and  hydurilic  acid ; 
while  the  second  is  represented  by  allantoin,  allantoic  acid,  and 
allituric  acid.  * 

The  best  known  representative  of  the  ureids  is  uric  acid.  From 
it  all  others  can  be  derived,  and  a  description  of  its  general  proper- 
ties and  reactions  may  serve  as  an  illustration  of  the  entire  class. 

Uric  acid  is  a  crystallizable  dibasic  acid  of  the  formula  C5H4N403. 
Structurally  it  may  be  regarded  as  a  purin  derivative,  and  may  be 
represented  by  the  formula  : 


HN— CO 

I      I 
CO   C— NHS 

!      Ii 

HN — O-NH' 


)CO. 


It  was  first  obtained  synthetically  by  treating  a  mixture  of  glycocoll 
and  urea  at  a  temperature  of  230°  C.  until  the  melted  mass  assumed 
a  yellowish  color  and  became  turbid.  The  reaction  which  took  place 
may  be  represented  by  the  equation  : 

CH2.NH2.COOH  +  3CO(NH2)2  =  C5H4N403  +  2H20  +  3NH3 
Glycocoll.  Urea.  Uric  acid. 

The  same  result  may  be  reached  by  heating  a  mixture  of  urea  and 
trichlorolactic  amide : 

C3H4C13N02  +  2C0(NH2)2  =  C5H4N403  +  H20  +  HC1  +  NH4C1 
Trichlorolactic  amide. 

As  indicated  in  the  previous  section,  moreover,  uric  acid  also  results 
through  the  condensation  of  isodialuric  acid  and  urea,  according  to 
the  equation  : 

HN— CO  HN-CO 

II  II 

CO  C  +  CO(NH2)2  =     CO  C— NH. 

||  |       ||  >CO  +  2H20 

HN— C(OH)  HN— C— NH^ 

When  treated  in  the  dry  state  uric  acid  is  decomposed  into  hydro- 
cyanic acid,  cyanuric  acid,  ammonium  carbonate,  ammonium  cyanate, 
biuret,  etc.  Fused  with  an  excess  of  potassium  hydrate,  it  similarly 
yields  ammonia,  potassium  carbonate,  oxalate,  cyanate,  and  cyanide. 

On  reduction  with  nascent  hydrogen,  in  the  presence  of  water  and 


THE   UREIDS.  83 

sodium  amalgam  uric  acid  is  first  transformed  into  xanthin,  and  sub- 
sequently into  hypoxanthin.  The  close  relationship  which  exists 
between  the  nucleinic  bases  and  uric  acid  is  thus  further  shown 
(see  page  80). 

Analogous  to  the  synthetic  formation  of  uric  acid  from  urea  and 
glvcocoll,  we  find  that  on  decomposition  with  hvdriodic  acid  the 
substance  yields  carbon  dioxide,  ammonia,  and  glycocoll,  viz.,  the 
same  products  which  are  obtained  from  the  nucleinic  bases. 

From  the  reactions  which  have  thus  far  been  described  the  nature 
of  uric  acid  as  a  ureid  is  not  apparent.  If  the  hydrolytic  decom- 
position of  the  substance  is  effected  less  energetically,  this  becomes 
manifest  at  once.  On  prolonged  boiling  with  water  it  is  decom- 
posed into  urea  and  dialuric  acid,  which  -latter  further  yields  urea 
and  tartronic  acid.  In  this  manner  the  character  of  uric  acid  as  a 
diureid  of  the  first  order  is  demonstrated.  The  reactions  which 
take  place  are  represented  by  the  equations : 

(1 )  C5H4NA  +  2H20  =  04H4NA  +  CO(NH^a 

Uric  acid.  Dialuric  acid.         Urea. 

(2)  C,HtX,04  +  2H20  =  C3HA       +  CO(NH2)2 
Dialuric  acid.  Tartronic  acid.  Urea. 

On  treating  with  an  oxidizing  agent,  in  the  presence  of  water,  uric 
acid  is  similarly  decomposed  into  urea  and  the  mono-ureid  alloxan, 
which  can  be  further  decomposed  into  urea  and  mesoxalic  acid  : 

(1)  C5H4N,03  +  H20  +  O  =  C4H2N204  +  CO(NH2)2 
Uric  acid.  Alloxan. 

(2)  €4H2X.A  +  H20  =  C3HA      +  COfNH,), 

Alloxan.  Mesoxalic 

acid. 

Its  relation  to  the  di-ureids  of  the  second  order  is  shown  by  oxidiz- 
ing the  substance  with  peroxide  of  manganese  in  neutral  solution  at 
a  moderate  temperature.  In  this  manner  allantoin  is  formed,  from 
which,  on  further  oxidation,  urea  and  oxalic  acid  result: 

(1)  C5H4X403  +  H20    +  O  =  qftNA  +  C02 

Allantoin. 

(2)  C4H6X403  +  2H20  +  O  =  C2HA  +  2CO(NH2)2 

Oxalic 
acid. 

Of  special  interest,  further,  is  the  formation  of  murexid,  or 
ammonium  pur put-ate,  which  results  when  uric  acid,  even  in  minimal 
amount--,  is  evaporated  together  with  nitric  acid,  and  the  reddish 
r<  sidue  i-  brought  in  contact  with  ammonia.  A  beautiful  purplish- 
red  color  then  develops,  which  is  characteristic  of  uric  acid  and  its 
-alt-  (murexid  test).  The  reactions  which  take  place  maybe  rep- 
resented by  the  equations : 

I)    c.H.NA  +2H20        =(',ir,NA  +CO(NH2)2 

Dialuric  acid. 
(2)  r.H.N'A  -f-NH4OH   =C4H8(NH4)NA  +  HSQ 

\m  111'. niiim 
(liillur.it' 

(?,,    2CIII./\H ,)N20(  +  0  =('.]|/\|[,).\<)1;    I    311,0 

Ammonium  dialurate.  Murexid. 


84      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

The  relation  of  uric  acid  to  the  mono-ureids  of  the  second  order, 
finally,  is  shown  by  treating  one  part  of  uric  acid  with  three  parts 
of  nitric  acid  (50  per  cent,  solution),  and  heating  to  70°  C.  On  sub- 
sequent evaporation  to  a  syrup  and  cooling,  parabanic  acid  crystal- 
lizes out,  and  on  decomposition  yields  urea  and  oxalic  acid  : 

(1)  C5H4N403  +  H20  +  O  =  C4H2N204  +  CO  (NH,), 

Alloxan. 

(2)  C4H2N204  +  O  =  C3H2N203  +  C02 

Parabanic 
acid. 

(3)  C3H2N203  +  2H20         =  C2H204      +  CO(NH2)2 

Oxalic  acid. 

Guanin,  as  has  been  shown,  under  the  same  conditions  yields 
guanidin  and  parabanic  acid,  which  further  illustrates  the  close 
relationship  between  the  two  classes. 

For  a  consideration  of  the  methods  employed  in  the  isolation  of 
uric  acid  and  its  quantitative  estimation,  the  reader  is  referred  to  the 
chapter  on  the  Urine. 

THE   KREATINS. 

The  kreatins,  or  kreatinic  leucomains,  as  they  are  also  termed  by 
Gautier,  are  basic  substances  which  are  closely  related  to  the 
nucleinic  bases  and  to  the  ureids,  which  have  just  been  considered. 
They  comprise  kreatin,  kreatinin,  crusokreatinin,  xanthokreatinin, 
amphikreatinin,  and  two  similar  substances  of  doubtful  composition. 
Kreatin,  moreover,  is  related  to  arginin,  and  can  be  produced  syntheti- 
cally through  the  union  of  cyanamide  and  methyl-glycocoll,  as  arginin 
results  from  cyanamide  and  ornithin.  While  arginin,  however,  can 
be  obtained  artificially  from  albuminous  material,  kreatin  has  thus 
far  not  been  isolated  in  this  manner.  Its  formation  from  cyanamide 
and  methyl-glycocoll  is  represented  by  the  equation : 

/NH2 
N  =  C  — NH2  +  NH(CH3)  —  CH2—  COOH  =  NH  =  C< 

XN(CH3)  —  CH2— COOH 
Cyanamide.  Methyl-glycocoll.  Kreatin. 

It  is  thus  a  homologue  of  glycocyamin,  which  results  through  the 
union  of  cyanamide  and  glycocoll : 

/NH2 
N  =  C  —  NH2  +  CH2  — NH2  —  COOH  =  NH2  =  C< 

\NH  — CH2-COOH 
Cyanamide.  Glycocoll.  Glucocyamin. 

On  dehydration  kreatin  loses  one  molecule  of  water  and  is  trans- 
formed into  kreatinin,  which  may  thus  be  regarded  as  the  anhydride 
of  kreatin  : 

XNH2  .NH  CO 

NH  =  C<  =  NH  =  C(  +  H20 

XN(CH3)  —  CH2  —  COOH    '  NN(CHS)  — CH, 


THE  AMIDO- ACIDS.  85 

On  oxidation  with  mercuric  oxide  kreatin  gives  rise  to  the  forma- 
tion of  the  oxalate  of  methyl-guanidin  or  methyl-urarnin,  which  in 
turn  results  from  guanidin,  and  this  from  guanin. 

-ClII9X302  +  50  =  (C,H7X3)2.C,H204  +  2C02  +  H20 
Kreatin.'  Methyl-guanidin 

oxalate. 

On  decomposition  with  baryta-water  kreatin  yields  urea,  methyl- 
glycocollj  and  a  small  amount  of  hydantoic  acid.  Kreatinin  is 
similarly  transformed  into  methyl-hydantoin,  which  is  then  likewise 
decomposed  into  urea  and  methyl-glycocoll,  thus  demonstrating 
the  close  relationship  which  exists  between  the  kreatins  and  the 
ureid-. 

.NH2  CH2  —  NH.CH3 

NH  =  C{  +  H20  =   I  +  CO(NH2), 

NN(CH3)  —  CH2  —  COOH  COOH  Urea. 

Kreatin.  Methyl-glycocoll. 

/XH CO  /NH CO 

NH  =  C  I      +H20  =  NH3  +  CO<  I 

X(CH3)  —  CH2  XN(CH3)  —  CH2 

Kreatinin.  Methyl-hydantoin. 

Kreatin  and  kreatinin  are  homologous  with  lysatin  and  lysatinin, 
which,  as  has  been  seen,  result  from  albuminous  material  when  this 
is  decomposed  with  boiling  mineral  acids.  While  kreatin,  however, 
contains  but  four  atoms  of  carbon,  lysatin  contains  six,  and  is 
represented  by  the  formula  C6H13N302.  Like  kreatin  and  kreatinin, 
lysatin  and  lysatinin  yield  urea  on  hydrolytic  decomposition. 

The  individual  representatives  of  the  group  will  be  considered 
later. 

THE   AMIDO-ACIDS. 

The  amido-acids  represent  one  of  the  most  important  groups  of 
chemical  substances  which  are  found  in  the  animal  and  the  vegetable 
world.  They  are  intimately  concerned  in  the  construction  of  the 
albuminous  molecule,  and  are  accordingly  always  found  among  the 
decomposition-products  of  all  albumins.  Some  of  them  belong  to 
the  BO-called  diamido-acids,  while  others  are  mono-amido-acids. 
With  the  latter  we  shall  deal  more  exclusively  at  this  place.  They 
are  generally  of  the  character  of  a  fatty  acid  or  of  an  aromatic  acid, 
in  which  one  hydrogen  atom  of  the  group  CH8  or  C,.H5  has  been 
replaced  by  the  amido-radicle  NHj.  Acetic  acid  thus  gives  rise  to 
tnono-amido-acetic  acid,  and  benzoic  acid  to  mono-amido-benzoic 
acid,  etc,  a-  shown  by  the  formulae: 

CHg.COOH,  acetic  acid. 

CHj  MI,  1.'  noil,  mono-amido-acetic  acid. 

I  ,11  '  « 1' ill.  benzoic  add. 

I  ,,l I, '  \  1 1 1   <  K )OH,  mono-amidobenzoic  acid. 


86      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

The  most  important  members  of  the  group  which  will  here  be 
considered  are  glycocoll,  leucin,  asparaginic  acid,  glutaminic  acid, 
and  tyrosin.     They  are  represented  by  the  formulae  : 

CH2.(NH2).COOH,  glycocoll. 

(CH3)2.CH2.CH(NH2).COOH,  leucin. 

CH2.CH(NH2).(COOH)2,  asparaginic  acid. 

(CH2)2.CH(NH2).(COOH)2,  glutaminic  acid. 

C6H4(OHJ.CH2.CH(NH2).COOH,  tyrosin. 

It  will  thus  be  seen  that  glycocoll  and  leucin  are  amido-deriva- 
tives  of  the  mono-basic  acids  of  the  formic  series,  viz.,  amido-acetic 
acid  and  a-amido-capronic  acid,  while  asparaginic  acid  and  glutam- 
inic acid  are  dibasic  acids  of  the  oxalic  series,  viz.,  amido-succinic 
and  amido-glutaric  acids.  Tyrosin,  on  the  other  hand,  is  an  amido- 
derivative  of  the  aromatic  series,  viz.,  para-oxy-a-amido-propionic 
acid. 

They  are  all  derivatives  of  the  albumins,  and,  as  has  been  indi- 
cated, integral  constituents  of  the  albuminous  molecule.  Their 
quantitative  relations,  however,  are  subject  to  considerable  variation, 
and  from  some  albumins  indeed  not  all  can  be  obtained.  Tyrosin, 
for  example,  is  lacking  in  glutin  and  collagen,  and  glycocoll  is 
similarly  not  found  in  casein.  In  some  leucin  predominates,  while 
in  others  tyrosin  stands  in  the  foreground.  These  variations  will 
be  considered  in  greater  detail  when  we  shall  deal  with  the  various 
digestive  products  of  the  albumins. 

The  amido-acids  of  the  fatty  series  are  of  special  interest  to  the 
physiological  chemist,  owing  to  the  fact  that  they  are  apparently 
intimately  concerned  in  the  production  of  urea.  Von  Schroder, 
Nencki,  and  others  have  thus  shown  that  in  the  liver  the  ammonium 
salt  of  carbamic  acid,  viz.,  amido-formic  acid,  is  transformed  into 
urea,  and  we  also  know  that  in  the  mammalian  organism  leucin, 
glycocoll,  and  asparaginic  acid  are  likewise  transformed  into  urea 
and  eliminated  as  such.  That  a  portion  of  the  urea,  indeed,  origin- 
ates in  this  manner  can  scarcely  be  doubted.  As  regards  the 
nature  of  the  chemical  changes  which  take  place  during  the  trans- 
formation of  the  amido-acids  into  urea,  our  knowledge  is  not 
complete.  It  was  formerly  supposed  that  uric  acid  represented  the 
immediate  antecedent  of  urea  and  was  transformed  into  this  by 
oxidation.  We  find,  as  a  matter  of  fact,  that  in  birds  and  reptiles 
uric  acid  constitutes  the  final  decomposition-product  of  the  nitro- 
genous metabolism,  and  is  thus  analogous  to  the  urea  of  mammals. 
I  have  also  pointed  out  that  as  a  ureid,  uric  acid  on  oxidation 
can  yield  urea.  But  as  far  as  is  known,  the  uric  acid  of  mammals  is 
exclusively  derived  from  the  nucleinic  bases,  and  is  thus  scarcely 
found  in  sufficient  quantity  to  give  rise  to  the  large  amount  of  urea 
which  is  daily  eliminated  in  the  urine.     That  a  small  fraction  of 


THE  AMIDO-ACIDS.  87 

the  urea  may  result  from  uric  acid  by  simple  oxidation  is  possible, 
and  indeed  probable,  but  the  greater  portion  must  of  necessity 
originate  in  a  different   manner. 

In  birds,  on  the  other  hand,  some  of  the  uric  acid  apparently 
results  from  glycocoll,  and  we  thus  see  that  in  both  classes  of  animals 
the  amido-acids  may  be  the  antecedents  of  the  final  products  of 
nitrogenous  metabolism. 

It  is  conceivable  that  in  mammals  cyanic  acid  may  be  produced 
as  an  intermediary  product,  and  that  urea  then  results  through  a 
condensation  of  two  molecules  of  this  substance  in  statu  nascendi, 
according  to  the  equation  : 

/NH2 
COXH  +  CONH  =  CO<  +  C02. 

XNH2 

Then  again  we  may  imagine  that  a  transformation  of  the  amido- 
acids  occurs  into  the  ammonium  salts  of  the  fatty  acids  standing 
next  in  order  in  the  downward  scale,  and  that  by  further  oxidation 
these  are  transformed  into  ammonium  carbonate,  and  this  into  urea. 
It  has  been  shown  as  a  matter  of  fact  that  a  fair  amount  of  urea  is 
thus  produced  when  blood  containing  ammonium  carbonate  or  ammo- 
nium formate  is  allowed  to  flow  through  the  isolated  livers  of  dogs. 
According  to  Drechsel,  finally,  the  amido-acids  are  transformed 
into  carbamic  acid,  from  which  urea  may  then  result,  as  indicated  by 
the  equation  : 

/NH2  XNH2  /NH, 

CO<  +  CO<  =  CO<  +  C02  +  H20. 

OH  xOH  XH2 

In  a  subsequent  chapter  this  subject  will  be  treated  at  greater  detail. 

In  addition  to  the  important  rdle  which  the  amido-acids  thus 
play  in  the  formation  of  urea,  these  bodies  are  of  further  interest 
from  the  part  which  they  take  in  some  of  the  syntheses  that 
occur  in  the  animal  organism.  In  this  manner  they  give  rise  to  a 
Dumber  'if  complex  substances  which  can  hence  be  regarded  as 
amido-derivatives.  With  benzoic  acid  glycocoll  thus  combines  to 
form  hippuric  acid,  as  shown  by  the  equation: 

<  II     MI   i.OOOB       C6H5.COOB       MI  ,.MI<  <  JU'Oi.COOII       II,0 
Glycocoll.  Benzoic  acid.  Hippuric  acid. 

With  phenyl-acetic  acid  glycocoll  similarly  combines  to  form 
phenaceturic  acid  : 

rii,.(XH,> .moil       mi     <    ii    .COOH 

CeHj.CHj.CO  —  NH.CHj.COOH       II.o. 

That  uric  acid  on  bydrolytic  decomposition  will  yield  ammonia, 

carbon  dioxide,  and  glycocol]    hag    been  shown.      There    is   evidence, 

moreover,  to  show  thai  in  the  organism  of  birds  and  reptiles,  at 
least,  it-  synthesis  can  similarly  occur. 

Ornithuric  acid  results  through  the  union  of  benzoic  acid  with  the 
diamide  ornithin  : 


88      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

C4H7(NH2)2.COOH  +  2C6H5.COOH  =  C4H7(NH.C6H5.CO).,.COOH  +  2H20 
Ornithin.  Benzoic  acid.  Hippuric  acid. 

The  amido-acids  also  are  closely  related  to  the  biliary  acids,  as  on 
decomposition  with  baryta-water,  glycocholic  acid  is  decomposed 
into  glycocoll  and  cholalic  acid,  as  shown  in  the  equation  : 

C26H43N06  +  H20  =  CH2(NH2).COOH  +  C24H40O5 

Glycocholic  Glycocoll.  Cholalic 

acid.  acid. 

Taurocholic  acid  similarly  gives  rise  to  cholalic  acid  and  taurin, 
which  latter  can  be  regarded  as  amido-isethionic  acid — that  is,  as 
isethionic  acid,  H(C2H4.OH)S03,  in  which  the  hydroxyl  group 
has  been  replaced  by  the  amido-radicle  : 

C26H45N07S  +  H20  =  C24H40O5  +  C2H7N03S 
Taurocholic  Cholalic  Taurin. 

acid.  acid. 

After  the  ingestion  of  amido-acids,  moreover,  the  corresponding 
compounds  of  carbamide,  — CONH,  appear  in  the  urine.  These  are 
spoken  of  as  the  uramic  acids,  and  comprise  methyl-hydantoinic  acid, 
taurocarbamic  acid,  uramido-benzoic  acid,  and  tyrosin-hydantoinic 
acid,  or  hydantoin-hydroparacumaric  acid.  They  are  found  after 
the  ingestion  of  sarcosin  or  methyl-glycocoll,  of  taurin,  amido- 
benzoic  acid,  and  tyrosin,  respectively.  The  syntheses  which  are 
thus  effected  may  be  represented  by  the  equations  : 

C3H702      +   CONH  =  C4H8N203 
Sarcosin.  Methyl-hydantoinic 

acid. 

C2H7NS03  +   CONH  =  C,H8N2S04 
Taurin.  Taurocarbamic 

acid. 

C7H7N02     +   CONH  =  C8H8N203 

Amidobenzoic  Uramido-benzoic 

acid.  acid. 

C9HHN02   +    CONH  =  CipH10N2O3   +    H20 
Tyrosin.  Tyrosin  hydantoinic 

acid. 

Among  the  mono-amido  acids,  finally,  which  may  be  formed  in 
the  animal  body,  we  must  mention  cystein,  or  a-amido-thiolactic 
acid.  This  is  of  special  interest,  as  its  disulphide — cystin — is  occa- 
sionally found  in  human  urine,  and  is  then  commonly  associated 
with  the  diamins  putrescin  and  cadaverin.  The  relation  which 
exists  between  cystin  and  cystein  is  thus  similar  to  that  of  a  mer- 
captan  to  its  disulphide,  and  may  be  represented  by  the  equation  : 

H3C  v       /SH  H3C  \       /S S\       /CH3 

2  >C<  +0=      ^     >C(  >C<  +H20 

h2n/    xcooh  h2n/    xCOOH    COOH'    xnh2 

Cystein.  Cystin. 

Analogous  to  the  synthetic  formation  of  hippuric  acid  after  the 
ingestion  of  benzoic  acid,  we  find  in  dogs  and  rabbits  that  following 
the  administration  of  mono-bromobenzol  bromophenyl-mercapturic 


THE  AMIDO-ACIDS.  89 

acid  appears  in  the  urine.     The  reaction  which  here  takes  place  may 
be  represented  by  the  equation  : 

HoC  \        /oil 

\c<  -    CJi5Br-CH2(XH2).COOH  = 

H2N        XCOOH  GlycocoU. 

Cvbteiu.  H,C\       /S.CGH4.Br 

>C<  +  NH3  +  2H20 

CH^.CO.NH/     \COOH 

Bromophenyl-mereapturic 
acid. 

On  reduction  theamido-acids  are  transformed  into  the  correspond- 
ing acids  from  which  they  are  derived.  Glycocoll  is  thus  transformed 
into  acetic  acid,  leucin  into  capronic  acid,  asparaginic  acid  into  suc- 
cinic acid,  glntaminic  acid  into  glutaric  acid,  tyrosin  into  para-oxy- 
phenyl-propionic  acid  (hydroparacumaric  acid),  etc.,  as  shown  by 
the  equations  : 

CH2(XH,).COOH  +  2H  =  CH3.COOH  +  NH3 

Glycocoll.  Acetic  acid. 

/COOH  /COOH 

CH,.CH(NH,)/  +  2H  =  CH2.CH2.<  +  NH3 

"       COOH  XCOOH 

Asparaginic  acid.  Succinic  acid. 

XCH  /OH 

C6H4  +2H  =  C6H4/  '  +NH, 

\CH2.CH(NH2).COOH  xCH2.CH2.COOH 

Tyrosin.  Para-oxy-phenyl-propionic 

acid. 

On  oxidation  these  are  further  changed  into  the  acids  standing 
next  in  order  in  the  downward  scale.  Acetic  acid  thus  gives  rise  to 
the  formation  of  formic  acid,  succinic  acid  to  malonic  acid,  para-oxy- 
phenyl-propionic  acid  to  para-oxy-phenyl-acetic  acid,  etc.,  as  shown 
by  the  equations : 

CH3.COOH  +  30  =  H.COOH  +  H20  +  C02 
Acetic  acid.  Formic  acid. 

/COOH  /COOH 

CH./H,.  +  30  =  CH,  +  H20  +  C02 

COOH  xCOOH 

Succinic  acid.  Malonic  acid. 

OH  /OH 

C6ir,  +30  =  CBH1  +  H20  +  CO2 

CHj.CHj.COOH  CHj.COOH 

Para-oxy-phenyl-propionic  Para-oxy-plieiiyl-acetic 

acid.  acid. 

Through  a  splitting  off  of  carbon  dioxide  para-oxy-phenyl-acetic 
acid  theu  further  gives  rise  to  paracresol,  from  which  phenol  is 
finally  obtained  on  oxidation  : 

OB  OH 

(,!l,  =C6H4<         +C02 

I  II ..ex )H  OH, 

ParacresoL 

oil 
I  ,11,  +30  =  (',,11, OH  -f  CO, +  H,0 

<  |[  PhenoL 


90      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

In  the  animal  body  paracresol  and  phenol,  which  are  formed  from 
tyrosin  during  the  process  of  intestinal  putrefaction,  then  combine 
with  sulphuric  acid,  and  are  eliminated  through  the  urine  in  the 
form  of  their  potassium  salts : 

/OH  .OH  /O.HSO3 

C6HZ         +  S02<         =C6H4<  +H20. 

XCH3  \OH  \CH3 

/OH 
C6H5.OH      +  S02<         =  QH.O.HSO,     +  H20. 
XOH 

During  the  process  of  albuminous  putrefaction  other  aromatic 
compounds  besides  tyrosin  and  its  derivatives  also  result,  but  these, 
in  contradistinction  to  tyrosin,  belong  to  the  ortho-group.  They 
comprise  indol,  skatol,  and  skatol-carbonic  acid.  According  to  some 
observers,  these  substances  are  derived  from  a  special  aromatic  radicle, 
which  is  contained  in  the  albuminous  molecule,  and  which  differs 
from  the  tyrosin  radicle,  while  others  believe  that  they  result  syn- 
thetically through  the  influence  of  the  living  bacteria  from  certain 
aromatic  groups,  which  on  hydrolytic  decomposition  with  acids  give 
rise  to  tyrosin. 

Indol,  skatol,  and  skatol-carbonic  acid  are  closely  related  to  each 
other  and  to  indigo.  Skatol  thus  results  from  indol  through  a  sub- 
stitution of  the  methyl-group  for  a  hydrogen  atom  of  one  of  the  CH 
groups,  as  shown  by  the  formula  : 

/CH  ^  /C(CH3)<v 

c6h/      Jck  c6h4<        J^ch 

Indol.  Skatol. 

Skatol-carbonic  acid  then  results  from  skatol  through  a  union  with 
carbon  dioxide : 

/C(CH3)a. 
C6H/  3CC00H 

Skatol-carbonic  acid. 

In  their  passage  through  the  animal  body  indol  and  skatol  are 
oxidized  to  indoxyl  and  skatoxyl,  and  are  eliminated  in  the  urine  to 
a  great  extent,  in  combination  with  sulphuric  acid,  as  potassium 
indoxyl  sulphate  (animal  indican)  and  potassium  skatoxyl  sulphate, 
while  the  skatol-carbonic  acid  is  excreted  as  such.  These  changes 
can  be  expressed  by  the  equations  : 

/CH.  /C(OHU 

(1)  C6H /        >CH  +0  =  C6H /  J^CH 

\NH/  \NH^^ 

Indol.  Indoxyl. 

/C(OH)  .  ^C(O.SOsH)< 

(2)  C6H4<  ^CH  +  H2SO,  =  C6H4< 

Indoxyl. 


THE  PTO MAINS.  91 

On  decomposition  with  strong  hydrochloric  acid  indican  is  accord- 
ingly decomposed  into  sulphuric  acid  and  indoxyl,  which  latter  can 
then  be  oxidized  to  indigo-blue  : 

/C(OH)  ^  /CO  x  /CO  x 

2C6H/  >CH  +  20  =  C6H4<         >C  =  C<         >C6H4  +  2H20 

Indoxyl.  Indigo-blue. 

On  reduction  indigo-blue  is  transformed  into  indigo-white, 
C8H6NO,  which,  when  boiled  with  zinc  and  water,  then  further 
yields  indol : 

C8H5NO  +  H    =  C8H6NO. 

C8H6NO  +  3H  r-  C8H7N  +  H20. 

Animal  indican,  however,  must  not  be  confused  with  vegetable 
indican,  which  is  a  glucoside,  and  yields  indigo-blue  and  indiglucin 
on  hydrolytic  decomposition : 

C26HS1N017  +  2H20  =  C8H5NO  +  3C6H10O6 
Vegetable  indican.  Indigo-blue.      Indiglucin. 

A  small  amount  of  skatoxyl,  indoxyl,  and  phenol  is  also  eliminated 
in  the  urine,  in  combination  with  glucuronic  acid,  as  skatoxyl, 
indoxyl,  and  phenol  glucuronates,  respectively.  This  acid  may  be 
derived  from  glucose  by  the  substitution  of  one  atom  of  oxygen  for 
two  atoms  of  hydrogen,  and  is  accordingly  represented  by  the 
formula  COOH.(CH.OH)4.COH.  It  is  possible,  however,  that  glu- 
curonic acid  may  also  be  derived  from  chondroitin-sulphuric  acid, 
which  is  normally  found  in  cartilage,  and,  as  a  matter  of  fact,  we 
have  seen  that  through  a  series  of  simple  reactions  glucuronic  acid 
can  be  obtained  from  this  source.  On  oxidation  it  is  transformed 
into  saccharinic  acid,  the  relation  of  which  to  glucose  has  already 
been  considered. 

THE  PTOMAINS. 

The  term  ptomain  was  originally  applied  by  Selmi  to  certain 
alkaloidal  bodies  which  are  formed  during  the  process  of  albu- 
minous putrefaction.  Gautier  then  extended  its  use  to  include  all 
those  alkaloidal  substances  which  result  from  anaerobic  fermenta- 
tion, as  also  those  which  are  formed  in  the  tissues  of  the  higher 
animals  in  the  absence  of  air,  or  in  the  presence  at  least  of  an  insuf- 
ficient Bupplyof  oxygen.  In  contradistinction  to  these  substances, 
Gautier  terms  those  alkaloidal  bodies  which  are  formed  during  the 
normal  and  aerobic  life  of  the  tissues  leucomains.  Under  this 
latter  heading,  as  has  been  -ecu,  he  comprises  the  nucleinic  bases 
and  the  kreatins.  Both  classes  of  substances  are  of  special  interest 
to  the  physician,  as  their  formation  or  undue  accumulation  in  the 
body  may  give  rise  to  serious  disturbances.  This  is  true  more 
particularly  of  the  ptomains,  some  of  which  are  extremely  toxic. 

Gautier  divides  the  ptomains  into  acyclic  and  cyclic  ptomains, 


92      THE  NITROGENOUS  DERIVATIVES  OF  THE  ALBUMINS. 

some  of  which  are  free  from  oxygen,  while  in  others  this  is  present. 
They  are  all  derived  from  ammonia  by  the  substitution  of  various 
radicles  for  one  or  all  of  the  nitrogen  atoms  of  ammonia,  and  are 
hence  analogous  to  the  amins. 

The  acyclic  ptomains  which  are  free  from  oxygen  comprise  the 
following  substances : 

Methylamin NH2.CH3     =  CH5N 

Dimethylamin NH(CH3)2    =  C2H7N 

Trimethylamin N(CH3)3       =  CgHgN 

Butylainin ]STH2(C4H9)  =  C4HnN 

Amylamin NH2(C5Hn)  =  C5H13]Sr 

Hexylamin NH2(G6HI3)  =  C6H15N 

JSTeuridin C5HUN2 

Saprin      C5HUN2 

Pentamethylene-diaruin  or  cadaverin NH2(CH2)5.NH2 

Ethylene-diamin NH2(C2H4)4NH2 

Tetraniethylene-diamin  or  putrescin      NH2(CH2)4.NH2 

Dimethylene-imide  or  spermin (CH2)2NH 

Mydalein 

Metliyl-guanidin CH4(CH3)N3 

The  oxygen-containing  acyclic  ptomains  are  the  following : 

Cholin  or  trimethyl-oxyethylene-am-  1         N(CH3)3.(C2H4.OH).OH  =  C5HI5N02 
monium  hydrate J  v       i,A  *  °      °       i 

NhUvdrate  trimethy1-viny1"ammonium  }  N(CH3)3.(C2H3) .OH  =  C5H13NO 

Muscarin     '.  '.  '.  '.  '.  '.  '.    .    .  '.  '.  '.    .  N(CH3)3.(CH2.COH).OH  =  C5H15N03 

Betain  or  oxycholin       N(CH3)3.(CH2.COOH).OH  =  C5H,;N02 

Mvdatoxin C6H13N02 

Mydin      C8HnNO 

Gadinin C7HI6N02 

Methyl-gadinin       C8H18N02 

Mytilotoxin C6Hl5N02 

Propyl-glucocyamin C6H13N203 

The  remaining  ptornains  are  partly  cyclic  and  in  part  not  classified  : 

Collidin  (iso-phenyl-ethyiamin)  .    .    .    .  C6H5.CH(CH3).NH2  =  C8HnN 

Hydrocollidin C8H13N 

Parvolin C9H,3N 

Corindin C10H15N 

Hydrolutidin C7HHN 

Hydrocornidin C10H17N 

Scombrin      Cj7H38N4 

Morrhuin C19H27N3 

Asellin      C25H32N4 

Morrhuicacid C5H3(OH)(C3H6.COOH).NH  =  CqH13N03 

Typhotoxin C7H17N02 

Tetanin Ci3H30N2O4 

Tetanotoxin C5H,,N 

Spasmotoxin 

Tyrotoxin 

Pyocyanin 

Pyoxanthin 

Some  of  these  substances  and  their  origin  from  the  albumins  have 
already  been  considered,  and  we  shall  have  further  occasion  to  study 
them  in  greater  detail.  Others  are  scarcely  known,  and  require  no 
further  description  at  this  place.     They  are  all  intimately  related  to 


THE  ORGANIC  SOX-NITROGENOUS  ACIDS.  93 

the  vegetable  alkaloids,  with  which  they  have  many  reactions  in 
common.  They  have  strongly  basic  properties,  and  are  capable  of 
combining  with  acids  to  form  salts.  Like  the  albumins  from  which 
they  are  derived,  they  are  precipitated  with  the  chlorides  of  platinum, 
mercury,  and  gold,  as  also  with  tannic  acid,  picric  acid,  phospho- 
molybdic  acid,  phosphotungstic  acid,  etc.  With  these  they  form 
well-defined  crystalline  salts,  which  serve  for  their  differentiation 
from  each  other  and  as  a  basis  for  the  determination  of  their  ele- 
mentary composition.  The  methods  which  are  employed  for  the 
separation  of  ptomains  will  be  considered  in  a  subsequent  chapter. 

THE    ORGANIC   NON-NITROGENOUS   ACIDS. 

The  organic  non-nitrogenous  acids  which  are  formed  in  the  animal 
body  are  largely  members  of  the  tatty  acid  series.  Others  belong  to 
the  glycolic  series,  still  others  to  the  acrylic  series,  some  are  repre- 
sentatives of  the  oxalic  series,  and  still  others  belong  to  the  aromatic 
oxy-acids.  The  first  group  comprises  the  following  acids,  which  as 
a  class  may  be  represented  by  the  formula  C„H2n02. 

Formic  acid H.COOII  =  CH.,02 

Acetic  acid CH3.COOH  =  C2H402 

Propionic  acid CH,.CH3.COOH        =  C3H60„ 

Butvricacid (CH2)2.CH3.COOH   =  C4H802" 

Valerianic  acid (CH2)3.CH3.COOH    =  C5H10O2 

Capmnicacid (CH2)4.CH3.COOH   =  C6H„02 

Palmitic  acid (CH2)14.CH3:COOH  =  C16H320, 

Stearic  acid (CH2)16.CH3.COOH  =  C18H36(J2 

The  two  last,  as  has  been  seen,  are  integral  constituents  of  the 
fats,  in  which  they  are  present  in  combination  with  glycerin  as  tri- 
glycerides. From  these  the  others  may  in  part  be  derived,  but  to 
the  greatest  extent  no  doubt  they  result  from  the  amido-acids 
through  a  process  of  oxidation  or  reduction,  as  illustrated  by  the 
equations  : 

CH2(XII,).COOH  +  20  =  H.COOH  +  NH3  +  C02 
Glycocoll.  Formic 

acid. 

ny  nh2).cooh  +  2H  =  ch8.cooh  +  nh3 

Glycocoll.  Acetic  acid. 

Through  further  oxidation  these  acids  are  then  transformed  into 
those  standing  nexl  in  order  in  the  downward  scale,  and  so  on, 
until  finally  carbon  dioxide  and  water  result,  as  seen  in  the  equations  : 

fh   <  II,  (  Il.moH  +  30  =  CH,.COOH    |   C02  +  II,<> 
Propionic  acid.  Acetic  acid. 

(2)  CHg.COOH         +30  =  H.C00H      -(- C02  +  H20 
e  acid.  Formic 

acid. 

B.COOH  <)        OOj  |   U,p 

Formic  add. 

To  some  extent,  however,  the  fatty  acids  are  derived  also  from  lactic 
acid  and  related  acids,  which,  as  will  !><■  seen  later,  arc  constantly 


94      THE  NITROGENOUS  DERIVATIVES   OF  THE  ALBUMINS. 

being  formed  in  the  metabolism  of  the  albumins.  Their  transforma- 
tion into  the  fatty  acids  is  then  probably  analogous  to  the  production 
of  butyric  acid  during  the  process  of  butyric  acid  fermentation. 

2C3H603  =  C4H802  +  2C02  +  4H 
Lactic  Butyric 

acid.  acid. 

The  glycolic  acids  which  are  found  in  the  animal  body  may  be 
represented  by  the  general  formula  CnH2„03.  They  comprise  the 
following  bodies : 

Glycolic  acid CH2.OH.COOH  =  C.2H403 

Ethylidene-lactic  acid  (paralactic  acid)   .    CH3.CH(OH).COOH  =C3H603 

Ethylidene- lactic acid(optically inactive)  CH3.CH(OH).COOH  =C3H603 

Ethvlidene-lsevo-lacticacid CH3.CH(OH).COOH  =C,H603 

/3-oxybutvric  acid CH.OH.CH2.CH3.COOH  =  C,H803 

Leucinicacid (CH3)2.CH.CH2.CH.OH.COOH  =  C6H1203 

Of  these  acids,  glycolic  acid  does  not  occur  in  the  animal  body,  so 
far  as  is  known,  but  it  is  of  interest  owing  to  the  fact  that  it  is 
closely  related  to  glycocoll,  and  is  derived  from  this  in  the  same 
manner  in  which  leucinic  acid  is  obtained  from  leucin,  viz.,  by  the 
substitution  of  a  hydroxyl  group  for  the  amido-group,  as  shown  by 
the  equations : 

CH2.(NH2).COOH    +  H20  =  CH2(OH).COOH  +NH3 
Glycocoll.  Glycolic  acid. 

(CH3)2.CH.CH2.CH.(NH2).COOH  +  H20  = 

Leucin.  (CH3)2.CH.CH2.CH(OH).COOH  +  NH3 

Leucinic  acid. 

/3-oxybutyric  acid  is  found  only  under  pathologic  conditions. 

Lactic  acid  and  its  isomeric  compounds,  as  well  as  leucinic  acid 
and  /3-oxybutyric  acid,  are  all  albuminous  decomposition-products, 
and  in  part,  at  least,  derived  from  the  amido-acids.  The  origin  of 
lactic  acid,  however,  is  not  so  clear,  but  we  shall  consider  this  ques- 
tion in  greater  detail  in  a  subsequent  chapter. 

On  reduction  these  acids  can  be  transformed  into  fatty  acids. 
Lactic  acid,  as  has  just  been  shown,  thus  gives  rise  to  butyric  acid, 
leucinic  acid  is  similarly  changed  to  capronic  acid,  etc. 

On  oxidation  /3-oxybutyric  acid  is  transformed  into  diacetic  acid, 
which  in  turn  is  decomposed,  with  the  formation  of  acetone  and 
carbon  dioxide : 

CH,.CH.OH.CH2.COOH  +  O  =  (CH3.CO)CH2COOH  +  H20 
J3-oxybutyric  acid.  Diacetic  acid. 

(CH3.CO)CH2.COOH  =  CO(CH3)2  +  C02 
Acetone. 

The  bodies  which  are  thus  formed  are  of  special  interest  to  the 
pathologist,  as  their  accumulation  in  the  animal  body  is  apparently 
capable  of  causing  very  serious  disturbances.  Acetone,  however,  is 
also  met  with  under  normal  conditions,  and  apparently  represents  a 
constant  product  of  albuminous  decomposition. 

On  boiling  with  dilute  mineral  acids  /3-oxybutyric  acid  is  trans- 


THE  ORGANIC  NON-NITROGENOUS  ACIDS.  95 

formed  into  an  acid  of  the  acrylic  series  (see  below),  viz.,  a-crotonic 

acid : 

CH3.CH(OH).CH2.COOH  =  2H20  +  CH3  —  CH  =  CH.COOH 

a-Capronie  acid. 

The. acids  of  the  acrylic  series  can  be  represented  by  the  general 
formula  CBH2„_202.  Representatives  of  these  are  the  a-capronic 
acid,  just  referred  to,  and  oleic  acid,  which  as  a  triglyceride  rep- 
resents a  must  important  constituent  of  many  of  the  vegetable  and 
animal  fats.  On  heating  with  hydriodic  acid  and  red  phosphorus  to 
a  temperature  of  210°  C,  oleic  acid  takes  up  two  atoms  of  hydro- 
gen, and  is  thus  reduced  to  stearic  acid  : 

CH3.  ( 'II , |, ,CH  =  CH.CH2.COOH  +  2H  =  CH3.(CH2)16.COOH 
Oleic  acid,  Stearic  acid. 

On  treating  with  nitrous  acid,  in  statu  nascendi,  it  is  transformed 
into  the  solid  elaidinic  acid,  which  is  isomeric  with  oleic  acid  and 
belongs  to  the  same  series. 

The  dibasic  acids  of  the  oxalic  series  can  be  represented  by  the 
formula  C„H2n_204.  They  include  oxalic  acid,  succinic  acid,  and 
glutaric  acid,  of  which  the  two  latter  are  generally  met  with  in  the 
form  of  their  amides,  viz.,  as  asparaginic  acid  and  glutaminic  acid, 
respectively.  In  this  form  they  are  invariably  obtained,  together 
with  oxalic  acid,  as  decomposition-products  of  most  albumins. 
Oxalic  acid  and  succinic  acid,  however,  may  also  be  observed  as 
such. 

The  relation  of  oxalic  acid  to  the  ureids  has  already  been  con- 
sidered.    They  are  represented  by  the  formulae : 

Oxalic  acid (COOH)2  =  C2H204 

Succinic  acid      (CH2)2.(COOH)2  =  C4H*<  >< 

Glutaric  acid (CH2)3.((X)OH)2  =  C5H8G4 

On  oxidation  they  are  decomposed  into  water  and  carbon  dioxide, 
but  it  is  probable  that  during  this  transformation  fatty  acids  are 
formed  as  intermediary  products : 

(  JI204  =  C02  +  H.COOH. 

C4HA  =  C02  +  C3H602 
Propionic 

acid. 

The  principal  aromatic  oxy-acids  which  may  be  found  in  the 
animal  body  are  hydroparacumaric  acid  or  para-oxy-phenyl-pro- 
pionic  acid,  para-oxy-phenyl-acetic  acid,  para-oxy-plienyl-lactic 
acid  or  oxy-hydroparacumaric  acid,  and  para-oxy-phenyl-glycolic 
acid.  They  are  derivatives  of  phenol,  in  which  one  hydrogen  atom 
has  been  replaced  by  tin-  radicle  of  the  corresponding  non-aromatic 
acid,  a-  shown  by  the  equation  : 

on 
r  If  oil      CH..CH.OH.COOH      0      CJ3L  +  II20 

ooL  actic  acid.  (II. (II  CH.COOH 

oxy-phenyl- 
Lactic  acid. 


96      THE  NITROGENOUS  DERIVATIVES   OF  THE  ALBUMINS. 

They  are  probably  all  derivatives  of  tyrosin,  and  it  has  already 
been  shown  (p.  89)  how  hydroparacu marie  acid  results  from  this  on 
reduction,  and  is  then  transformed  into  para-oxy-phenyl-acetic  acid 
by  oxidation. 

The  formulae  of  these  acids  and  their  relation  to  tyrosin  are  seen 

below : 

.OH 
Tyrosin  (para-oxy-phenyl-amido-propionic  acid)  =  C6H4<^ 

xCH2.CH(NH2).COOH 

X)H 

Para-oxy-phenyl-propionic  acid  =  C6H4<( 

\CH2.CH2.COOH 

.OH 

Para-oxy-phenyl-acetic  acid        =  C6H4<f 

XCH2.COOH 

,OH 

Para-oxy-phenyl-acetic  acid        =  CfiH4<; 

XCH9.C00H 


.OH 
Para-oxy-phenyl-lactic  acid        =  C6H/ 


Para-oxy-phenyl-glycolic  acid     =  CgH^ 


CH2.CH.OH.COOH 

OH 

CHOH.COOH 


On  decomposition  these  acids  yield  phenol  or  cresol,  water,  and 
carbon  dioxide,  as  shown  by  the  equations : 

.OH  /OH 

(1)  C6H /  +  30  =  C6H4<  '  +  C02  +  H20. 

\CH2.CH2.COOH  \CH2.COOH 

.OH  /OH 

(2)  C6H/  +  30  =  C6H4<  +  C02  +  H20 

\CH2.COOH  \COOH 

Para-oxy- 
benzoic  acid. 

/OH 

(3)  C6H /  =  C6H5.OH  +  C02 

\COOH  Phenol. 

or 

/OH  /OH 

(1)  C6H/  =  C6H/         +  C02 

\CH2.COOH  XCH3 

Paracresol. 

/OH 

(2)  C6H  /         +  30    =  C6H5.OH  +  C02  +  H20 

\CH3  Phenol. 

From  phenol  the  two  dioxybenzols  pyrocatechin  and  hydro- 
quinon  can  then  result,  and  appear  in  the  urine  together  with 
phenol  as  conjugate  sulphates  or  glucuronates. 

/0H(1) 
C6H5.OH  +  O  =  C6H4< 
6    5  X0H(2)  or  (4). 


THE  ORGANIC  NON-NITROGENOUS  ACIDS.  97 

Of  the  non-hydroxvlated  aromatic  acids,  two  are  found  in  tlie 
animal  body,  and,  like  the  hvdroxylated  compounds,  originate  during 
the  process  of  albuminous  putrefaction.  These  are  phenyl-propionic 
acid  (hydroeinnamic  acid)  and  phenyl-acetic  acid.  They  are  rep- 
resented by  the  formulae : 

C6H5  CPLj.CBLj.COOH  =  phenyl-propionic  acid 
C6H5.CII2  COOH  =  phenyl-acetic  acid. 

By  combining  with  glycocoll  they  give  rise  to  the  formation  of 
hippuric  acid  and  phenaceturic  acid,  respectively.  In  the  first 
instance,  however,  benzoic  acid  is  apparently  first  formed,  which 
then  unites  with  glycocoll,  as  shown  in  the  equations  below : 

(1)  C6H5.CH2.CH,.COOH  +  60  =  C6H3.COOH  +  2C02  +  2H20 

Phenyl-propionic  Benzoic  acid, 

acid. 

(2)  C6HvCOOH  +  CH2(NH2).C00H        =  C6H5.CO.CH2.NH.COOH  +  H20 
Benzoic  acid.  Glycocoll.  Hippuric  acid. 

and 

C6H5.CH2.C00H  +  CH2(NH2).COOH  =  C6H5.CH,.CO.NH.CH3.CO0H  +  H20 
Phenyl-acetic  Phenaceturic  acid, 

acid. 


CHAPTEK    VI. 

THE  FERMENTS. 

In  the  foregoing  chapters  we  have  considered  in  a  general  way 
the  more  important  characteristics  of  the  three  great  classes  of 
food-stuffs,  and  have  studied  in  some  detail  also  the  various  decom- 
position-products to  which  they  give  rise  in  their  passage  through 
the  animal  body.  We  have  also  pointed  out  that  with  few  excep- 
tions the  food-stuffs,  which  the  animal  derives  either  directly  or 
indirectly  from  the  plant,  cannot  be  utilized  by  the  animal  directly, 
but  that  they  must  previously  undergo  certain  changes,  which  vary 
with  the  character  of  the  individual  substances.  The  native  albu- 
mins must  first  be  transformed  into  albumoses  and  peptones ;  the 
disaccharides  and  polysaccharides  must  be  inverted  to  monosac- 
charides, and  the  fats  must  first  be  emulsified.  We  have  seen  also 
that  in  the  chemical  laboratory  these  changes  can,  for  the  most  part, 
be  brought  about  through  the  action  of  superheated  steam,  by  boiling 
with  acids  and  alkalies,  etc. — that  is,  through  agencies  which  mani- 
festly are  not  at  work  in  the  living  world.  The  question  therefore 
suggests  itself :  What  are  the  means  at  the  disposal  of  living  animals 
to  bring  about  these  changes  ?  This  question  has,  in  a  measure, 
been  answered  in  the  introductory  remarks,  where  it  was  pointed 
out  that  the  animal  is  capable  of  bringing  about  a  large  number  of 
analytical  changes  by  means  of  certain  ferments,  or  enzymes,  which 
are  furnished  by  the  animal  cells  themselves.  At  the  same  time  it 
was  pointed  out  that  the  better  known  representatives  of  this  class 
are  essentially  hydrolytic  ferments,  but  that  there  is  evidence  also  of 
the  existence  of  oxidation-ferments. 

As  the  chemical  processes  which  take  place  in  the  animal  body 
are  essentially  of  the  character  of  hydrations  and  oxidations,  it 
would  thus  appear  that  other  factors  besides  the  ferments  would 
be  unnecessary  for  the  functioning  of  the  various  organs.  It  is 
quite  possible,  indeed,  that  this  is  actually  the  same,  and  that  the 
various  manifestations  of  life  may  be  explained  upon  the  basis  of 
fermentative  phenomena.  It  must  be  admitted,  however,  that  with 
the  exception  of  those  ferments  which  are  at  work  in  the  gastro- 
intestinal canal,  and  through  the  agency  of  which  the  ingested  food- 
stuffs are  transformed  into  substances  that  can  be  utilized  by  the 
body,  our  knowledge  of  such  ferments  is  extremely  limited  ;  and  we 
are  scarcely  in  a  position,  as  yet,  to  state  definitely  that  all  those 
chemical  processes  which  take  place  in  the  living  animal  are  brought 
about  in  this  manner. 


THE  FERMENTS.  99 

We  do  not  even  know  in  what  manner  the  transformation  of 
albumoses  and  peptones  into  native  albumins  is  etfected,  although  we 
have  abundant  evidence  that  this  transformation  takes  place  in  the 
epithelial  cells  which  line  the  gastro-intestinal  canal.  The  existence 
of  oxidation-ferments  in  the  tissues,  further,  is  even  denied  by  very 
capable  observers.  Then,  again,  we  have  no  direct  evidence  that  cer- 
tain synthetic  processes  which  occur  in  the  animal  body,  such  as  the 
formation  of  fats  from  carbohydrates,  are  brought  about  through  the 
agency  of  ferments;  although  in  plants,  as  we  have  seen,  this  may 
in  all  likelihood  occur.  On  the  other  hand,  we  know  that  many 
ferments  exert  their  special  activity  within  the  bodies  of  the  cells, 
and  are  not  secreted  to  the  outside  ;  and  that  for  this  reason  they  are 
iu  a  measure  removed  from  observation.  As  a  matter  of  fact,  such 
ferments  have  been  isolated  from  certain  micro-organisms,  and  have 
been  shown  to  be  capable  of  manifesting  a  certain  activity  even  after 
the  death  of  the  mother-cells.  Experiments,  however,  which  have 
been  undertaken  for  the  purpose  of  obtaining  such  ferments  from 
the  animal  tissues  have,  on  the  whole,  not  yielded  encouraging 
results.  Nevertheless,  it  must  be  confessed  that,  as  our  knowledge 
of  the  ferments  is  as  yet  very  incomplete,  future  investigations  may 
still  show  that  the  so-called  vital  forces  are  in  reality  the  forces 
which  are  characteristic  of  ferments  or  related  bodies;  and,  as  has 
been  pointed  out,  these  forces  are  essentially  the  same  as  those  which 
we  meet  with  in  the  non-organized  world. 

In  the  present  chapter  we  shall  deal  in  greater  detail  with  the 
ferments  as  a  class.  The  ferments  proper  must  be  sharply  distin- 
guished from  the  so-called  ferment-organisms,  or  organized  ferments, 
which  occur  widely  distributed  in  nature  and  comprise  the  impor- 
tant groups  of  bacteria,  blastomycetes,  and  certain  moulds.  These 
are  living  beings  in  themselves,  and  not,  as  the  ferments  proper, 
products  of  life.  They  contain  ferments,  and  manifest  their  special 
activity,  in  a  great  measure  at  least,  through  their  ferments  ;  but 
they  are  not  ferments  themselves,  although  they  are  often  so  called. 
In  contradistinction  to  these  organized  ferments,  the  ferments  proper 
are  also  termed  non-organized  ferments,  or  enzymes.  They  are 
ap  icific  products  of  the  activity  of  certain  cells,  and  occur  not  only 
in  the  animal,  but,  as  we  have  seen,  also  in  the  vegetable  world. 

The  activity  which  is  manifested  bv  the  non-organized  ferments, 
so  far  a-  we  can  isolate  them  from  their  mother-cells,  is  not  the  same, 
however,  ;i-  that  which  tlie  cell  can  exhibit  as  a  whole,  but  only  a 
part,  while,  on  the  other  hand,  the  cellular  activity  includes  that  of 
it-  ferment.  When  common  beer  yeast  is  thus  placed  in  a  solution 
of  cane-sugar  the  cell  is  not  only  capable,  through  its  ferment,  of 
invert  in-/  iln-  cane-sugar  into  glucose  and  Isevulose,  but  it  can  of 
itself  cause  tin-  further  destruction  of  these  sugars,  with  the  forma- 
tion of  aicohol  and  carbon  dioxide.  In  this  latter  process  the  fer- 
ment plays  ii"  part,  a-  can  be  readily  shown  by  placing  some  fresh 
yeast  in  water   to    which   a    certain   amount  of  chloroform   has  been 


100  THE  FERMENTS. 

added,  so  as  to  bring  about  the  death  of  the  cells  proper.  If  some 
of  this  liquid  is  now  added  to  a  solution  of  cane-sugar,  an  inversion 
takes  place,  as  before,  but  subsequent  fermentation  does  not  occur. 
In  the  first  experiment  we  thus  see  manifested  the  activity  of  the 
living  cell  as  such,  as  also  that  of  its  ferment ;  while  in  the  second 
test  only  that  of  the  ferment  is  shown.1 

For  the  maintenance  of  life,  in  the  case  of  the  higher  plants  at 
least,  the  organized  ferments  are  of  prime  importance ;  for,  as  has 
been  seen,  it  is  through  these  low  forms  of  life  that  the  higher  plants 
are  furnished  their  nitrogen  in  a  form  which  they  can  subsequently 
utilize.  In  their  absence  from  the  soil,  vegetable  life,  such  as  we  see 
it,  could  probably  not  exist.  In  the  gastro-intestinal  tract  of  all 
animals  which  have  been  examined  in  this  direction  innumerable 
bacteria  are  also  found,  and  it  was  long  thought  that  their  presence 
here  served  a  very  definite  purpose,  and  that  animal  life  could  not 
go  on  in  their  absence.  This  view,  however,  has  proved  erroneous, 
as  Nuttall  and  Thierfelder  conclusiyely  demonstrated.  They  showed 
that  when  a  young  guinea-pig,  for  example,  is  removed  from  the 
mother  animal  by  Cesarean  section  under  strict  aseptic  precautions, 
and  is  subsequently  fed  with  sterile  food  and  is  furnished  with  sterile 
air,  it  will  grow  as  well  as  a  control-animal.  While  the  presence  of 
bacteria  in  the  animal  body  is  therefore  not  essential  for  the  main- 
tenance of  life,  and  while  it  is  very  questionable,  indeed,  whether 
their  presence  in  the  alimentary  canal  serves  any  useful  purpose  at 
all,  we  know,  on  the  contrary,  that  the  introduction  of  certain  forms 
is  directly  harmful,  and  that  some  of  the  normal  inhabitants  of  the 
intestinal  canal  may  under  certain  conditions  develop  distinct  patho- 
genic properties. 

From  a  physiological  standpoint  the  organized  ferments  are  con- 
sequently of  secondary  interest  only  in  animal  chemistry,  while 
pathologically  they  may  be  most  important.  It  is  thus  known  that 
during  their  metabolism  they  can  give  rise  to  the  formation  of  sub- 
stances which  are  more  or  less  toxic,  and  which  when  absorbed  into 
the  blood  cause  definite  pathological  symptoms.  Such  bodies  are  the 
so-called  ptomains  and  toxalbumins.  The  former  are  basic  sub- 
stances which  belong  to  the  fatty  series,  and  consist  of  carbon, 
hydrogen,  and  nitrogen,  and  in  some  instances  also  of  oxygen 
(see  page  91). 

While  some  of  the  ptomains  are  apparently  harmless,  others  are 
exceedingly  poisonous,  and  these  last  are  accordingly  spoken  of  as 
toxins.  Representatives  of  the  former  group  are  cadaverin  and 
putrescin,  two  diamins,  which  are  respectively  pentamethylene  and 
tetramethylene  diamin,  while  typhotoxin  and  tetanin  belong  to  the 
latter  class.  The  toxalbumins,  on  the  other  hand,  are,  as  the  term 
indicates,  albuminous  substances,  which  in  part  at  least  belong  to 

!Buchner,  it  is  true,  has  of  late  claimed  to  have  succeeded  in  causing  complete 
fermentation  also  in  the  absence  of  the  living  cell,  but  his  results,  while  confirmed 
by  some,  are  not  as  yet  accepted  by  all. 


GENERAL  PROPERTIES  OF  THE  FERMENTS.  101 

the  albumoses,  while  others  are  apparently  globulins,  and  still  others 
peptone-like  bodies.  Whether  these  various  substances  are  pro- 
duced by  the  bacteria  themselves  or  through  the  agency  of  the 
contained  ferments  is  not  definitely  known,  but  it  is  more  than 
likely  that  the  latter  are  intimately  concerned  in  their  formation. 

The  enzymes  or  ferments  proper,  to  which  Ave  shall  now  return, 
are,  as  has  been  pointed  out,  specific  products  of  cellular  activity, 
and  are  for  the  most  part  formed  in  the  cell-bodies  from  pre-existing 
substances,  the  so-called  proenzyme*  or  zymogens.  In  the  peptic 
cells  of  the  stomach,  for  example,  the  specific  ferment  pepsin  does 
not  exist,  but  there  is  present  the  proenzyme  pepsinogen,  which  can 
be  transformed  into  pepsin  by  meaus  of  dilute  hydrochloric  acid. 
"Whether  this  rule  holds  good  for  all  ferments,  however,  we  cannot 
say,  and  in  the  case  of  those  ferments  which  are  not  secreted  to  the 
outside  we  are  not  in  a  position  to  put  the  question  to  the  test.  That 
ferments  exist  which  manifest  their  specific  activity  within  the  cell 
is  known.  Such  a  ferment  is  found  in  a  certain  bacterium,  the 
Micrococcus  urea.  If  this  organism  is  added  to  fresh  urine,  it  will 
gradually  bring  about  the  decomposition  of  the  urea  which  is  present, 
with  the  formation  of  ammonium  carbonate.  On  filtering  such  de- 
composing  urine  through  a  Chamberland  filter,  so  as  to  remove  the 
bacteria,  and  on  adding  a  portion  of  the  filtrate  to  fresh,  sterile 
urine,  no  change  is  brought  about.  This  shows  that  the  ferment  has 
not  passed  into  solution.  If,  on  the  other  hand,  decomposing  urine 
is  precipitated  with  alcohol,  and  the  bacteria  which  are  thus  thrown 
down  together  with  the  mineral  salts  are  now  killed  with  absolute 
alcohol  and  ether,  it  is  possible  to  extract  the  ferment  from  the 
dried  cell-bodies  ;  and  such  solutions,  even  when  filtered  with  the 
utmost  care,  will  bring  about  a  decomposition  of  urea  similar  to 
that  caused  by  the  bacteria  themselves. 

General  Properties  of  the  Ferments. — From  what  has  been 
Baid,  it  is  clear  that  the  ferments  are  capable  of  manifesting  their 
special  activity  even  after  the  death  of  their  mother-cells,  and  it  is 
noteworthy  that  a  great  many  substances  which  are  distinct  proto- 
plasmic poisons  do  not  interfere  with  the  ferments  themselves.  Such 
substances  are  chloroform,  ether,  thymol,  toluol,  salicylic  acid,  arseni- 
oue  acid,  sodium  fluoride,  boric  acid,  hydroxy lamin,  glycerin,  etc. 
in  the  Study  of  (lie  ferments  these  bodies  are  of  great  importance,  as 
we  are  thus  enabled  to  exclude  the  protoplasmic  activity  of  living 
cells,  and  to  determine  whether  certain  chemical  phenomena  which 
we  observe  in  the  tissues  of  the  body  are  referable  to  the  action  of 
ferments  or  not. 

Other  chemicals,  however,  not  only  cause  the  death  <>f  the  cells, 
but  also  arrest  or  annihilate  the  action  of  the  ferments.  Such  sub- 
stances are  the  bichloride  of  mercury,  carbolic  acid,  the  mineral 
acids,  and  to  a  less  marked  degree  other  metallic  salts,  as  also 
picric  acid,  tannic  acid, etc.  It  is  to  be  noted,  however,  that  one 
ferment,  ;it  Least,  viz.,  pep-in,  i-  not  destroyed  by  dilute  acids.     The 


102  THE  FERMENTS. 

activity  of  the  ferments  is  further  decreased  with  an  increase  of  their 
specific  products  beyond  a  certain  degree.  Absence  of  water  like- 
wise inhibits  the  action  of  the  ferments,  but  this  is  at  once  re- 
established when  the  necessary  degree  of  moisture  is  supplied,  and 
it  is  possible  therefore  to  preserve  the  ferments  in  the  dry  state. 
During  the  process  of  drying,  however,  care  must  be  had  that  the 
temperature  does  not  exceed  a  certain  limit.  This  varies  with  the 
different  ferments,  but  it  may  be  stated  as  a  general  rule  that  all 
animal  ferments  are  killed  by  a  temperature  of  75°  C,  while  the 
vegetable  ferments  cannot  survive  a  temperature  of  80°  C.  In  the 
absence  of  moisture,  however,  they  can  apparently  withstand  much 
greater  heat,  and  it  is  said  that  dry  trypsin,  pepsin,  and  diastase  may 
be  heated  to  a  temperature  of  from  150°  to  160°  C  without  losing 
their  activity.  Strong  alcohol  destroys  the  action  of  certain  ferments, 
such  as  pepsin  and  diastase,  while  others,  like  the  fibrin-ferment,  are 
not  affected. 

The  most  peculiar  property  of  the  ferments,  and  the  one  which  is 
characteristic  of  all,  is  the  power  to  bring  about  an  amount  of 
chemical  change  which  is  out  of  all  proportion  to  the  quantity  of  the 
ferment  present,  while  the  ferment  itself  undergoes  no  apparent 
change.  The  common  pepsin  preparations  of  the  market  are  thus 
of  a  strength  that  1  part  by  weight  of  the  pepsin  will  digest  6000 
parts  by  weight  of  coagulated  egg-albumin,  and  Petit  claims  that  a 
preparation  from  his  laboratory  was  capable  of  dissolving  even 
500,000  times  its  weight  of  fibrin  in  seven  hours. 

That  the  ferments  themselves  undergo  no  change  while  exerting 
their  specific  action  can  be  readily  shown,  as  it  is  possible  to  re- 
obtain  them  from  the  various  digestive  mixtures  and  to  test  their 
efficacy  as  before. 

The  rapidity  with  which  the  action  of  ferments  takes  place  is 
often  most  remarkable,  and  is  especially  well  shown  during  the 
coagulation  of  milk   under  the  influence  of  chymosin. 

In  order  that  the  ferments  may  exhibit  their  activity  to  best 
advantage  a  definite  temperature  is  necessary,  which  varies  some- 
what with  the  different  ferments,  but  is  generally  about  that  of  the 
body.  Higher  as  well  as  lower  temperatures  gradually  inhibit  their 
action,  and,  as  has  been  seen,  destroy  it  entirely  when  75°-80°  C.  is 
reached.     Very  low  temperature  has  the  same  effect. 

The  presence  or  absence  of  oxygen  has  no  effect  upon  the  action 
of  ferments,  and  they  thus  show  a  distinct  difference  from  the 
organized  ferments,  which  are  more  or  less  dependent  upon  either 
its  presence  or  its   absence. 

The  reaction  of  the  medium  in  which  the  ferments  are  to  display 
their  activity  is  very  important,  and  varies  with  the  different  fer- 
ments. Some  of  these,  such  as  pepsin,  can  act  to  advantage  only 
in  an  acid  medium  ;  while  others,  such  as  ptyalin,  require  an  alka- 
line reaction  ;  and  still  others  can  act  in  acid,  alkaline,  and  neutral 
media,  but  exhibit  certain  preferences. 


CHEMICAL    COMPOSITION   AND    GENERAL   REACTIONS.     103 

In  conclusion,  the  reversible  action  of  ferments,  which  has 
recently  been  established,  must  be  briefly  considered.  It  has  long 
been  known  that  the  hydrolytic  decomposition  effected  by  ferments 
is  never  carried  to  an  end,  and  it  is  usually  stated  that  this  is  owing 
to  the  fact  that  a  gradual  increase  in  the  production  of  decom- 
position-products inhibits  the  action  of  the  ferments  in  question.  In 
the  light  of  more  recent  investigations,  however,  this  explanation  is 
not  satisfactory.  It  has  been  demonstrated  that  maltase,  when 
added  to  a  solution  of  maltose,  will  cause  inversion  of  the  latter  to 
glucose.  An  end-reaction  is  then  not  obtained  ;  but  if  this  solution 
is  now  added  to  a  solution  of  glucose  in  turn,  at  a  time  when  further 
inversion  does  not  occur,  it  will  be  noted  that  a  retransformation  of 
glucose  to  maltose  takes  place,  which,  however,  is  likewise  not 
complete.  It  thus  appears  that  the  ferment  is  not  only  capable  of 
causing  the  hydrolytic  decomposition,  but  also  the  synthesis  of  mal- 
tose ;  but  that  in  so  doing,  its  action  ceases  as  soon  as  a  certain 
equilibrium  of  reaction  has  been  established.  This  reversible  action 
cm  the  part  of  ferments  is,  of  course,  of  the  greatest  interest  to  the 
physiological  chemist,  in  showing  that  the  complex  syntheses  which 
take  place  in  plant-life  may,  to  a  certain  extent  at  least,  be  referable 
to  such  action,  and  to  forces  which  are  probably  also  at  work  in  the 
non-organized  world. 

Further  research  will  show  whether  this  action  is  common  to  all 
ferments. 

Chemical  Composition  and  General  Reactions. — Of  the  chem- 
ical composition  of  the  ferments  but  little  is  known  that  is  definite. 
This  is  owing  to  the  fact  that  isolation  of  any  one  of  the  ferments  in 
a  chemically  pure  form  has  thus  far  not  been  accomplished.  They 
apparently  contain  nitrogen,  and  are  usually  regarded  as  albumi- 
nous substances ;  but  it  is  still  a  matter  of  doubt  whether  this  is 
actually  the  case,  and  it  is  possible  that  the  supposition  of  their 
albuminous  nature  is  owing  to  their  being  contaminated  with 
albumins. 

Like  the  albumins,  they  are  as  a  class  non-diffusible.  They  are 
soluble  in  water,  and  can  be  precipitated  from  their  aqueous  solu- 
tions by  salting  with  ammonium  sulphate  or  by  the  addition  of 
strong  alcohol. 

When  kept  under  alcohol  for  any  length  of  time  some  of  the  fer- 
ments, such  as  pepsin  and  diastase,  are  rendered  inactive  and  are 
apparently  coagulated,  while  the  activity  of  others,  such  as  the 
fibrin  ferment,  remains  unaffected. 

Characteristic  general  reactions,  which  are  common  to  all  fer- 
ments, are  unknown.  Formerly  it  was  supposed  that  they  all  pos- 
sessed the  power  of  decomposing  hydrogen  peroxide,  but  it  appears 
that  this  property  does  not  belong  to  the  ferments  proper,  but  to 
adherent  particles  of  protoplasm.  Asa  matter  of  fact,  it  is  possible 
in  a  number  of  ferments  to  destroy  this  power  of  decomposing 
hydrogen  peroxide  without  influencing  their  specific  activity  in  the 


104  THE  FERMENTS. 

least.  If  pancreatic  juice  is  thus  heated  to  a  temperature  of  60°  C, 
and  then  allowed  to  cool  to  40°  C,  it  will  be  observed  that  the  fluid 
is  still  capable  of  digesting  albumins  and  of  inverting  starch,  while 
it  has  lost  the  power  of  decomposing  hydrogen  peroxide  entirely. 
Similar  results  may  be  obtained  on  heating  the  dry  ferments  to  a 
somewhat  higher  temperature,  by  treating  with  alcohol,  or  by  satu- 
rating their  solutions  with  neutral  salts. 

Mode  of  Action. — The  decompositions  which  the  ferments  are 
capable  of  effecting  in  suitable  media  are  essentially  of  a  hydrolytic 
character.  This  can  be  readily  shown  by  comparing  the  decom- 
position-products to  which  the  ferments  give  rise  with  the  original 
substances,  when  it  will  be  found  that,  practically  without  exception, 
the  former  contain  more  water.  Nasse,  moreover,  could  demon- 
strate a  distinct  increase  in  the  electrical  conductivity  of  watery 
solutions  of  starch,  for  example,  when  these  were  treated  with  dias- 
tase, showing  that  dissociated  molecules  of  water  must  have  been 
present.  As  to  the  manner,  however,  in  which  these  hydrolytic 
phenomena  are  brought  about  Ave  are  very  much  in  the  dark.  On 
the  one  hand,  we  may  suppose  that  the  molecular  oscillations  which 
take  place  in  the  molecules  of  the  ferments  are  of  such  a  nature 
as  to  bring  about  an  increase  in  the  molecular  oscillations  of  the 
substances  upon  which  the  ferments  exert  their  specific  activity,  and 
that  in  consequence  of  this  increase  in  the  oscillations  the  labile 
equilibrium  of  the  large  albuminous  or  polysaccharine  molecules  is 
disturbed,  which  in  turn  would  lead  to  new  combinations  of  atoms 
to  form  molecules  that  are  more  stable,  and  the  oscillations  of  which 
would  be  more  nearly  like  those  of  the  ferments.  According  to 
this  theory,  then,  the  action  of  the  ferments  would  be  what  has  been 
termed  a  katalytic  action,  and  analogous  to  the  katalytic  action  of 
various  metals,  such  as  platinum,  gold,  silver,  etc.,  which  in  fine 
suspension  behave  in  very  much  the  same  manner  'as  the  ferments 
(see  page  20).  On  the  other  hand,  we  may  suppose  that  the  action 
of  the  ferments  is  an  action  by-  contact,  such  that  one  molecule  of 
the  ferment  causes  hydrolytic  decomposition  of  one  molecule  of  an 
albuminous  substance,  for  example,  and  that  this  decomposition  in 
turn  causes  decomposition  of  the  adjacent  albuminous  molecules,  and 
so  on.  The  action  of  certain  ferments,  such  as  the  fibrin  ferment, 
and  that  which  causes  the  coagulation  of  milk,  might  very  well 
be  explained  upon  such  a  basis.  For  here  we  see  a  rapidity  of 
action  which  scarcely  admits  of  any  other  explanation  ;  and  direct 
contact  of  the  ferments  with  all  parts  of  the  surrounding  material, 
moreover,  is  excluded,  as  each,  ferment-molecule  must  of  necessity 
be  at  once  surrounded  by  a  layer  of  the  coagulated  albumin. 

Classification. — It  has  been  pointed  out  that  there  are  no  general 
reactions  which  are  characteristic  of  all  ferments.  The  ferments 
can  be  separated  into  groups,  however,  which  are  fairly  well  char- 
acterized   through  their   specific   activity    and   the    decomposition- 


CLASSIFICATION.  105 

products  to  which  they  gave  rise.  They  are  accordingly  divided 
into  the  following  classes  : 

1.  The  Proteolytic  Ferments. — These  comprise  the  animal  ferments 
pepsin  and  trypsin,  various  vegetable  ferments  such  as  papayotin, 
and  others  which  may  be  obtained  from  germinating  seeds  of 
Lupinus  angustifoleus,  Lupinus  luteus,  Vicia  Faba  and  Ricinus* 
major,  and  certain  ferments  which  may  be  obtained  from  bacteria. 
They  all  digest  the  various  albumins,  with  the  formation  of  albu- 
naoses,  and  some  of  them  also  cause  the  further  decomposition  of 
these  to  amido-acids,  hexon  bases,  etc. 

2.  The  Amylolytic  Ferments. — These  include  the  ptyalin  of  the 
saliva  and  the  diastatie  ferment  of  the  pancreatic  juice,  the  so-called 
vegetable  diastase,  and  related  ferments,  which  may  be  obtained 
from  bacteria.  Some  of  these  only  render  starch  soluble,  while 
others  carry  the  hydrolysis  further  to  the  formation  of  monosac- 
charide. 

3.  The  Inverting  Ferments. — These  are  apparently  closely  related 
to  the  amylolytic  ferments,  and  are  to  a  certain  extent  identical  with 
them.  They  invert  the  disaccharides  to  monosaccharides,  and, 
according  to  their  specific  effect  upon  cane-sugar,  maltose,  and 
lactose,  arc  termed  invertins,  maltases,  and  lactases,  respectively. 
Such  ferments  are  found  in  the  saliva,  the  pancreatic  juice,  and  the 
intestinal  juice,  in  many  of  the  higher  plants,  and  also  in  numerous 
organized  ferments. 

4.  The  Steatolytic  Ferments. — Such  ferments  cause  decomposition 
of  fats  into  glycerin  and  fatty  acids.  Representatives  of  this  order 
arc  the  so-called  steapsin  of  the  pancreatic  juice,  and  analogous  fer- 
ments that  have  been  found  in  the  vegetable  world,  notably  in  the 
<cj'A^  of  ricinus,  Papaver  somniferum,  Cannabis  sativa,  in  linseed, 
and  in  corn. 

5.  The  Coagulating  Ferments. — These  include  the  fibrin  ferment 
which  brings  about  coagulation  of  the  blood  ;  the  milk-curdling  fer- 
ment chymosin,  which  is  found  in  the  gastric  and  pancreatic  juice, 
and  a  hypothetical  ferment  which  is  thought  to  cause  the  coagula- 
tion of  myosin. 

6.  The  ferments  which  cause  decomposition  of  urea.  Such  fer- 
ments are  formed  by  a  large  number  of  micro-organisms,  such  as 
the  Micrococcus  urese,  the  Bacterium  urese,  the  Bacillus  fluores- 
ces, etc. 

7.  Ferments  which  cause  decomposition  of  giucosides.     These  are 

principally  found  in  the  higher  plants,  and  include  the  emulsin  or 
synaptose  of  bitter  almonds;  the  myrosin  of  mustard  seeds  and 
other  <  'ruciferae,  etc. 

It  will  be  noted  that,  with  the  exception  of  the  coagulating  fer- 
ment-. ;ill  otlnr  animal  ferments  that  have  thus  far  been  mentioned 
are  ferments  which  are  secreted  by  the  digestive  glands,  and  have, 
BO    far   a-    i-    known,  only    to   do    with    the  digestion    of  food-studs. 

They  are  without  exception  hydrolytic  ferments.     Of  ferments,  on 


106  THE  FERMENTS. 

the  other  hand,  that  are  capable  of  effecting  the  various  hydrations 
and  oxidations  which  take  place  in  the  tissues  of  the  body,  we  have 
not  made  mention.  As  a  matter  of  fact,  it  must  be  admitted  that 
with  very  few  exceptions  we  have  no  knowledge  as  yet  of  the 
existence  of  such  ferments.  I  say,  "  with  very  few  exceptions,"  for 
there  are  a  few  ferments  which  have  been  obtained  from  the  tissues, 
and  which  are  certainly  not  identical  with  the  known  digestive 
ferments.  For  the  sake  'of  convenience,  I  shall,  for  the  present, 
speak  of  these  as  : 

8.  The  Tissue-ferments. — Members  belonging  to  this  group  have 
been  found  in  the  liver,  the  kidneys,  the  spleen,  the  adrenal  glands, 
the  muscles,  etc.,  and  have  long  been  regarded  as  identical  with  the 
digestive  ferments.  Some  of  them,  no  doubt,  are  closely  related  to 
these,  and,  like  them,  capable  of  bringing  about  hydrolytic  decom- 
position of  albumins  and  carbohydrates.  Others,  however,  are  dis- 
tinctly different  in  being  essentially  oxidizing  ferments.  From  recent 
studies  of  these  oxidizing  ferments  it  appears  that  different  varieties 
exist.  One  of  them,  the  so-called  aldehydase  of  the  liver,  has  been 
more  carefully  studied,  and  will  be  considered  in  greater  detail  later. 

Of  special  interest  is  a  ferment  which  has  likewise  been  obtained 
from  the  liver,  and  which  is  capable  of  transforming  the  closely 
combined  nitrogen  of  albumins  into  amido-nitrogen,  and  of  splitting 
this  off  in  the  form  of  ammonia. 

From  what  has  been  said,  it  is  clear  that  our  knowledge  of  the 
agencies  which  are  at  the  disposal  of  the  animal  body  in  order  to 
effect  those  chemical  changes  that  are  necessary  for  the  maintenance 
of  life  is  very  imperfect.  That  ferments  bring  about  transforma- 
tion of  the  native  food-stuffs  into  chemical  bodies  which  can  be 
assimilated,  and  subsequently  rebuilt  into  tissues,  we  know.  That 
other  ferments  exist  which  can  cause  destruction  of  the  organized 
tissues,  with  the  formation  of  substances  that  can  be  readily  removed 
from  the  body,  is  extremely  probable.  But  whether  all  the  vital 
manifestations  of  the  animal  tissues  can  be  reduced  to  the  activity 
of  ferments,  we  do  not  know.  It  has  been  pointed  out  that  living 
cells  possess  the  power  of  causing  chemical  changes  which  differ 
from  those  that  are  effected  by  their  contained  ferments,  and  the 
question  naturally  suggests  itself,  To  what  extent  are  the  kata- 
bolic  phenomena  which  we  observe  in  the  animal  body  referable  to 
pure  protoplasmic  activity,  as  compared  to  the  action  of  ferments  ? 
This  question,  however,  we  are  not  yet  prepared  to  answer.  We 
know  that  living  protoplasm  is  capable  of  causing  oxidation  of  non- 
living matter,  but  we  do  not  know  in  what  manner  this  is  brought 
about.  Possibly  this  power  is  referable  to  the  presence  in  the  cell 
of  ferments  which  may  yet  be  isolated,  and  which  may  manifest 
their  activity,  like  that  of  the  other  ferments,  even  after  the  death 
of  the  mother-cell ;  but  it  is  also  possible  that  this  power  depends 
upon  the  presenoe  in  the  cell  of  combinations  of  atoms  which  cannot 


CLA  SSIFICA  TION.  107 

be  split  off  from  the  protoplasmic  molecule  without  being  themselves 
destroyed.  In  that  event  we  would  be  forced  to  believe  in  the  exist- 
ence of  a  vital  principle  unlike  the  forces  which  are  at  work  in  the 
non-organized  world,  and  a  principle  which  is  transmitted  from  the 
parent  to  its  offspring  in  the  ovum  and  spermatozoon.  For  the 
present  Ave  are  unable  to  offer  even  a  hypothesis  as  answer  to  such 
questions. 


CHAPTER  VII. 

THE  DIGESTIVE   FLUIDS. 

As  I  have  pointed  out,  the  greater  portion  of  the  food-stuffs  which 
are  ingested  by  animals  cannot  be  utilized  as  such  directly,  but  must 
first"  be  transformed  into  material  that  is  capable  of  diffusing  through 
animal  membranes.  These  changes  occur  in  the  gastro-intestinal 
tract,  and  are  effected  by  the  secretions  of  the  various  digestive 
glands,  viz.,  the  saliva,  the  gastric  juice,  the  pancreatic  juice,  the 
succus  entericus,  and  the  bile. 

THE  SALIVA. 

General  Characteristics. — The  saliva  is  the  secretory  product 
of  the  salivary  glands,  viz.,  the  parotid,  the  submaxillary,  and  the 
sublingual  glands,  to  which  the  secretion  of  the  smaller  mucous 
glands  of  the  oral  cavity  is  further  added. 

The  saliva  is  a  colorless,  inodorous,  tasteless,  somewhat  stringy 
and  frothy,  opalescent  fluid,  which  normally  possesses  a  slightly  alka- 
line reaction  and  a  specific  gravity  ranging  between  1.002  and  1.008. 
A  slightly  acid  reaction  may,  however,  also  be  observed,  and  is  then 
referable  to  the  presence  of  lactic  acid,  which  is  formed  through  the 
activity  of  micro-organisms,  from  food-material  that  has  gathered 
between  the  teeth  or  from  desquamated  epithelial  cells.  For  this 
reason  also  we  find  an  acid  reaction  of  the  mouth-cavity  on  rising 
in  the  morning. 

On  microscopical  examination  the  saliva  is  seen  to  contain  a 
variable  number  of  pavement  epithelial  cells  and  so-called  salivary 
corpuscles.  These  are  identical  with  the  mucous  corpuscles,  which 
are  found  in  all  mucous  membranes,  and  represent  young  leucocytes 
that  have  not  entered  the  blood-current.  They  are  derived  from  the 
lymph-follicles  of  the  mucous  membrane,  and  in  the  case  of  the 
saliva,  no  doubt,  to  a  great  extent  from  the  tonsils.  In  addition 
we  find  innumerable  bacteria,  and  at  times  also  schizomycetes  and 
moulds.  On  standing,  the  liquid  becomes  turbid,  owing  to  pre- 
cipitation of  calcium  carbonate,  which  frequently  also  forms  a  fine, 
iridescent  film  on  the  surface.  This  phenomenon  is  due  to  the 
escape  of  carbon  dioxide  from  the  saliva,  and  explains  the  formation 
of  tartar  on  the  teeth,  as  also  the  origin  of  the  somewhat  uncommon 
salivary  concretions  in  the  larger  ducts  of  the  glands. 

Amount. — The  amount  of  saliva  that  is  secreted  in  the  twenty- 
four  hours  varies  somewhat  even  in  health,  but  probably  does  not 

108 


THE  SALIVA.  109 

exceed  1500  c.c.  It  depends  upon  the  amount  of  nutriment  in- 
gested, the  act  of  chewing,  the  character  of  the  food,  the  mental 
condition,  etc.  Fright  may  arrest  its  flow  entirely.  After  the 
administration  of  pilocarpin,  or  during  the  inhalation  of  ether,  an 
abundant  secretion  of  saliva  occurs,  and  it  is  thus  possible  to  col- 
lect in  the  human  being  sufficient  quantities  for  analysis.  Atropin 
acts  in  the  opposite  manner,  and  can  arrest  the  flow  entirely. 

To  a  certain  extent  the  amount  secreted  is  dependent  upon  the 
blood-pressure,  but  it  does  not  follow  that  the  saliva  results  from  the 
blood-plasma  through  a  simple  process  of  filtration.  We  find  that 
in  the  submaxillary  gland,  for  example,  the  secretion  continues  for 
some  time  even  after  decapitation  of  the  animal.  The  secretory 
pressure,  moreover,  is  very  much  greater  than  the  blood-press- 
ure; and  after  the  administration  of  atropin,  which  paralyzes  the 
secretory  nerves,  we  further  find  that  while  electrical  stimulation 
of  the  chorda  calls  forth  an  increased  circulation  in  the  gland,  a 
secretion  of  saliva  does  not  occur.  These  experiments  show  that 
the  secretion  of  the  saliva  cannot  be  referable  to  a  simple  process 
of  filtration,  but  must  depend  upon  a  special  secretory  activity  on 
the  part  of  the  alveolar  cells.  We  thus  also  find  that  the  salivary 
glands  are  capable  of  eliminating  certain  chemical  substances,  such 
as  bromide-;  and  iodides,  from  the  body,  while  others,  like  iron  com- 
pounds, for  example,  are  not  removed  through  this  channel,  if  we 
disregard  the  trace  which  is  normally  present. 

Chemical  Composition. — The  chemical  composition  of  the  saliva 
is  qualitatively  fairly  constant.  Quantitative  variations,  however, 
are  common.  This  is  to  a  certain  extent  owing  to  the  fact  that  the 
different  glands  are  not  all  of  one  kind.  In  the  human  being  the 
parotids  are  thus  albuminous  glands,  the  sublinguals  mucous  glands, 
while  the  submaxillary  glands  furnish  a  mixed  secretion.  The 
character  of  the  secretion,  moreover,  may  vary  with  one  and  the 
same  gland.  The  salivary  glands  all  have  a  double  nerve-supply, 
which  is  partly  of  cerebral  origin  and  partly  derived  from  the  sym- 
pathetic system,  and  as  the  one  or  the  other  set  of  fibres  exercises 
its  stimulating  effect,  the  composition  of  the  individual  secretions 
will  vary.  In  the  submaxillary  gland  of  the  dog,  in  which  these 
relations  have  been  especially  studied,  on  stimulation  of  the  sym- 
pathetic fibre-  ;i  secretion  is  furnished  which  is  less  abundant,  but 
contains  a  larger  amount  of  solids,  than  the  secretion  obtained  on 
stimulation  of  the  chorda.  This  is  well  shown  in  the  following 
table,  which  i-  taken  from  Kiiline: 

pathetic  saliva.  Chorda  saliva. 

Specific  gravity     .   .   .  1.007-1.018  1.004-1.006 

Solids 16-18  pro  ruille  12-14  pro  mille 

On  dividhi'_r  all  the    nerve-    which    supply  the    salivary  gland-,  or 

following  the  administration  of  curare,  the  secretion  still  continues 
for  a  while,  bm  tin-  saliva  which  is  thus  furnished  contains  Bcarcely 
any  -olid  material,  and  i-  termed  paralytic  saliva. 


110  THE  DIGESTIVE  FLUIDS. 

Qualitatively,  as  has  just  been  stated,  the  normal  mixed  saliva  is 
of  fairly  constant  composition.  The  quantitative  variations  which 
may  occur  in  health  are  seen  from  the  following  analyses  of  human 
saliva,  which  are  taken  from  Hammarsten  : 

Frerichs.  Berzelius.        Hammerbacher. 

Water 994.1  992.9  994.2 

Solids 5.9  7.1  5.8 

Mucus  and  epithelium 2.13  1.3  2.2 

Soluble  organic  matter 1.42  3.8  1.4 

Inorganic  salts 2.19  1.9  2.2 

Potassium  sulphocyanide  ....      0.10  .    .  0.04 

An  analysis  of  the  inorganic  salts,  moreover,  calculated  for  1000 
parts  by  weight  of  mineral  ash,  gave  the  following  results  : 

Potassium     .    .        457.2 

Sodium 95.9 

Oxide  of  iron 50.11 

Oxide  of  magnesium 1.55 

Sulphuric  acid  (as  S03) 63.8 

Phosphoric  acid  (as  P205) 188.48 

Chlorine 183.52 

The  albumins  proper  of  the  saliva  are  said  to  be  similar  to  those 
of  the  blood-serum,  but  are  present  in  only  very  small  amount. 
They  may,  in  fact,  be  regarded  as  accidental  constituents,  as  the 
greater  portion  of  the  albumins  which  enter  into  the  composition  of 
the  glandular  cells  is  no  doubt  transformed  into  the  specific  secretory 
products  of  these  glands,  viz.,  into  mucin  and  the  amylolytic  fer- 
ment ptyalin.  In  the  cells  proper,  however,  these  substances  appar- 
ently do  not  exist  as  such,  but  as  mucinogen  and  ptyalingen,  which 
are  later  transformed  into  mucin  and  ptyalin,  respectively.  As  a 
matter  of  fact,  it  is  possible  to  obtain  the  inactive  ptyalinogen  from 
the  solids  of  the  horse,  and  to  transform  it  artificially  into  the  active 
ferment.  To  this  end,  it  is  only  necessary  to  collect  the  saliva  from 
the  parotid  gland  of  the  horse  under  antiseptic  precautions,  and  to 
prevent  the  further  access  of  micro-organisms.  A  secretion  is  thus 
obtained  which  is  perfectly  inert  when  brought  in  contact  with 
starch  solution,  while  a  corresponding  specimen  that  has  been  ex- 
posed to  the  air  at  once  begins  to  manifest  the  specific  activity  of 
free  ptyalin.  Of  the  manner  in  which  this  transformation  is  effected 
in  the  mouth,  we  are  as  yet  ignorant,  but  it  appears  that  the  bacteria 
which  are  here  normally  present  are  of  importance.  Similar  results 
are  reached  when  the  finely  hashed  glands  are  extracted  with  chloro- 
form-water, until  the  active  ferment  can  no  longer  be  obtained  in 
tin's  manner.  On  subsequent  treatment  with  a  very  dilute  solution 
of  acetic  acid  other  extracts  can  then  be  obtained,  which  are  as  active 
as  the  first,  thus  showing  that  a  substance  must  have  been  present 
which  could  not  be  isolated  with  the  chloroform-water,  but  which 
can  be  transformed  into  ptyalin  by  means  of  acetic  acid. 

Ptyalin. — The  ptyalin  or  salivary  diastase,  as  it  is  also  termed,  is 
an  amylolytic  ferment,  and  as  such  capable  of  causing  the  inversion  of 


THE  SALIVA.  Ill 

Btarch  to  sugar.  This  can  be  readily  demonstrated  as  follows  :  A  few 
cubic  centimeters  of  saliva  are  added  to  a  small  amount  of  starch  solu- 
tion and  kept  at  a  temperature  of  about  35°  C.  If  a  drop  of  this  mixt- 
ure is  then  tested  at  intervals  of  about  one  minute  with  a  dilute  solu- 
tion of  iodine,  it  will  be  observed  that  the  blue  color,  which  is  first 
obtained  by  bringing  a  drop  of  the  two  solutions  together,  soon  gives 
place  to  a  violet,  and  then  to  a  mahogany  brown,  and  that  still 
later  no  color-reaction  whatever  occurs.  As  soon  as  this  point  is 
reached  a  small  amount  of  the  starch  mixture  is  examined  with 
Trommer's  or  Fehling's  solution  (see  page  278),  when  the  presence 
of  sugar  can  be  established.  The  sugar  which  thus  results  is  maltose, 
whil(T the  intermediary  products  which  are  formed  during  the  inver- 
sion of  the  starch  are*  represented  by  erythrodextrin,  achroodextrin, 
and  isomaltose.  The  reaction  which  takes  place  may  be  represented 
by  the  equations : 

(1)  (C,.,H.,flOin),t  +    3H2O  =  3[(C1,HwOI0)17.C1,H22Ou] 
V            Amidulin  Erythrodextrin. 

(2)  3[(CY,H,0O10)17.O12H22Ou]  -f    6H2O  =  9[(C12H20()10)5.Cl2H22()n] 

(3)  9[((\MJ  >,„i5.C12H2A,]    +  45H20  =  o4C12H22Ou  =  54Cl2H22On 

Isomaltose.  Maltose. 

We  shall  return  to  these  reactions  in  the  next  chapter,  when  the 
digestion  of  the  food  is  considered  in  detail. 

To  isolate  the  ptyalin  from  saliva,  the  following  method,  which 
has  been  suggested  by  Gautier,  may  be  employed:  To  a  large 
quantity  of  saliva  (J8  per  cent,  alcohol  is  added  so  long  as  a  floccu- 
lent  precipitate  is  seen  to  form.  This  is  collected  on  a  small  filter 
and  dissolved  with  a  small  amount  of  distilled  water.  The  solu- 
tion is  treated  with  a  few  drops  of  a  solution  of  bichloride  of  mercury, 
in  order  to  remove  any  albuminous  material  that  may  be  present. 
In  the  filtrate  the  excess  of  the  bichloride  is  removed  with  hydrogen 
Bulphide,  when  the  remaining  liquid  is  evaporated  to  dryness  at  a 
temperature  not  exceeding  40°  C,  and  the  residue  is  treated  with 
Strong  alcohol.  The  insoluble  portion  is  then  dissolved  with  a  small 
amount  oi*  distilled  water,  filtered,  dialyzed  in  order  to  remove  inor- 
ganic -alts,  and  finally  precipitated  with  absolute  alcohol,  when  the 
ptyalin  will  separate  out  in  light  Hakes.  Obtained  in  this  manner, 
ptyalin  is  a  white  amorphous  substance,  which  is  soluble  in  water, 
dilute  alcohol,  and  glycerin.  In  neutral  or  slightly  alkaline  solu- 
tion, but  not  iii  acid  solution,  it  rapidly  transforms  boiled  starch 
into  sugar  at  a  temperature  of  from  36°  to  40°  C.  Heated  to  a 
temperature  of  60°  C.,  its  solutions  lose  this  power,  and  it  is  thus 
possible  to  distinguish  between  ptyalin  and  the  diastatic  ferment  of 
vegetable  origin,  tor  which  the  optimum  temperature  Lies  between 
60°  and  65°  C. 

Of  special  interesl  i-  the  feci  that  the  transformation  of  starch 
into  sugar  ceases  as  soon  as  the  latter  is  presenl  to  the  extent  oi 
from  2  to  2.5  per  cent.     This  phenomenon  is  common  to  all  eniy- 


112  THE  DIGESTIVE  FLUIDS. 

matic  processes,  and  is  probably  referable  to  the  establishment  of  a 
certain  equilibrium  of  reaction.  A  complete  transformation  of  the 
starch  could  occur  only  if  the  resulting  sugar  were  removed  as 
rapidly  as  it  is  formed.  So  long  as  it  is  present,  the  reversible 
action  of  the  enzyme  becomes  manifest,  and,  analogous  to  the  rever- 
sion of  glucose  to  maltose,  a  similar  retransformation  of  maltose  to 
dextrin  no  doubt  occurs. 

The  amount  of  ptyalin  which  is  secreted  in  the  twenty-four 
hours  has  not  been  determined.  Its  activity,  as  would  be  ex- 
pected, is  subject  to  considerable  variations.  It  is  greatest  in  the 
morning  on  rising,  and  then  steadily  diminishes  during  the  day. 
Immediately  before  meals,  however,  a  temporary  increase  is  ob- 
served, which  is  then  followed  by  a  marked  decrease. 

Of  the  chemical  nature  of  ptyalin  but  little  is  known.  Like  all 
other  ferments,  it  is  generally  regarded  as  an  albuminous  substance, 
and  on  the  application  of  dry  heat  it  develops  the  characteristic  odor 
of  burning  albumins.  It  is  nitrogenous,  but  does  not  give  the 
xanthoproteic  reaction.  From  its  solutions  it  can  be  precipitated 
with  acetate  and  subacetate  of  lead,  while  bichloride  of  mercury  and 
the  salts  of  platinum,  as  also  tannic  acid,  are  without  effect. 

In  the  human  being  ptyalin  is  formed  in  the  parotid  and  the 
submaxillary  glands  and,  as  will  be  seen  later,  also  in  the  pancreas, 
while  the  sublingual  glands  apparently  yield  no  ptyalin.  In  other 
animals  its  presence  in  the  saliva  is  variable.  In  the  typical 
carnivora  it  is  said  to  be  absent,  while  in  the  saliva  of  all  herbivor- 
ous auimals  it  is  uniformly  found. 

Of  other  ferments,  human  saliva  apparently  also  contains  traces 
of  maltase,  and  of  an  oxydase  of  unknown  character ;  invertin, 
however,  has  not  been  found.  Consequently  an  inversion  of  maltose 
to  glucose  may  also  take  place  in  the  early  stages  of  carbohydrate 
digestion,  but  is  certainly  insignificant  in  extent. 

The  digestive  importance  of  the  saliva  is,  in  man,  at  least,  but 
slight,  as  ptyalin  is  rapidly  destroyed  by  contact  with  the  acid 
gastric  juice.  During  the  process  of  mastication  and  deglutition, 
moreover,  it  has  scarcely  time  to  effect  much  change,  and  in  experi- 
ments in  vitro  we  find  that  the  amount  of  maltose  which  is  formed 
by  the  saliva  from  starch  is  small.  The  importance  of  the  salivary 
glands  as  digestive  glands  has  thus  been  much  overrated,  and  it  has 
been  conclusively  demonstrated  that  their  function  has  mostly  to  do 
with  the  preparation  of  the  food  for  the  act  of  deglutition.  This  is, 
of  course,  greatly  facilitated  by  its  thorough  lubrication  with  the 
mucus  that  is  furnished  by  the  salivary  glands,  and  which  in  reality 
represents  the  most  important  constituent  of  the  saliva. 

Mucin. — The  mucin  of  the  saliva  is  derived  from  the  submaxil- 
lary and  sublingual  glands,  as  also  from  the  small  mucous  glands 
which  are  found  imbedded  in  the  mucous  membrane  of  the  mouth. 
Its  formation  in  the  salivary  glands  is  apparently  under  the  control 
of  the  sympathetic  nervous  system,  as  it  is  secreted  in  much  larger 


THE  SALIVA.  113 

amounts  on  stimulation  of  these  fibres  than  of  the  corresponding 
cerebral  fibres.  According  to  Levene,  the  submaxillary  mucin  eon- 
tains  the  chondroitin-sulphuric  aeid  complex,  or  a  closely  allied 
group. 

To  the  presence  of  the  mucin  the  viscid,  stringy  character  of  the 
saliva  is  due.  The  substance  can  be  obtained  by  precipitation  with 
acetic  acid,  and  it  is  to  be  noted  that,  in  contradistinction  to  the 
mucin-like  substances  which  belong  to  the  class  of  the  nucleo-albu- 
mins,  the  precipitated  mucin  is  insoluble  in  an  excess- of  the  acid. 
In  dilute  solutions  of  the  alkalies  it  is  soluble,  and  it  is  thus 
possible  by  repeated  precipitation  and  solution  to  obtain  the  mucin 
in  fairly  pure  form.  In  alcohol  and  water  it  is  insoluble,  although 
in  the  latter  it  swells  to  form  a  jelly-like  material.  Unlike  the 
albumins,  it  is  not  coagulated  by  heat;  but,  like  these,  it  gives  the 
xanthoproteic  reaction,  the  biuret  reaction,  and  Millon's  reaction. 
It  contains  also  a  small  amount  of  sulphur.  On  boiling  with  dilute 
mineral  acids  mucin  is  decomposed  into  a  substance  which  resembles 
mid  albumin,  and  into  a  carbohydrate-like  body  which  reduces 
Fehlingr's  solution.  This  has  been  regarded  as  identical  with  Land- 
wehr's  animal  gum  ;  Hammarsten,  however,  states  that  from  the 
mucin  of  the  submaxillary  gland  a  gum-like  substance  is  obtained 
which  contains  nitrogen.  On  decomposition  with  strong  mineral 
acids  mucin  yields  leucin,  tyrosin,  and  hevulinic  acid  (see  also  page 
45).  In  the  dry  state  it  occurs  as  a  white  or  yellowish-gray 
powder. 

Within  the  cells  mucin  exists  as  so-called  mu&nog&ri,  which  prob- 
ably represents  a  compound  of  mucin  with  an  additional  albuminous 
substance. 

Sulphocyanides. — Traces  of  sodium  sulphoeyanide  are  in  man 
usually  found  in  every  specimen  of  normal  saliva.  It  is  secreted 
by  all  the  salivary  glands,  but  in  largest  amount  by  the  parotids. 
In  other  animals  its  presence  is  not  so  constant,  and  in  some  indeed 
it  i-  not  found.      In  man  also  it  is  at  times  absent. 

To  demonstrate  the  presence  of  sulphocyanides,  it  usually  suffices 
to  treat  a  few  cubic  centimeters  of  saliva,  which  have  been  slightly 
acidified  with  hydrochloric  acid,  with  a  few  drops  of  a  very  dilute 
solution  of  perchloride  of  iron,  when  a  red  color  will  be  seen  to 
develop.  If  no  residt  is  obtained  in  this  manner,  a  larger  quantity, 
such  as  10()  c.C  IS  evaporated  to  a  small  volume  and  tested  as 
described. 

Nitrites. — Small     amounts     of    nitrites    may    also    at    times    be 

observed,  and  are  no  doubt  derived  from  the  nitrates  ingested.  To 
tesf  for  tli<-<-,  ;i|)()iit  lo  <•.<•.  of  saliva  are  treated  with  a  few  drops 
of  [lasvay'e  reagent,  and  beated  t<»  a  temperature  of  80°  C,  when 
in  the  presence  of  nitrite-  ;i  red  color  develops. 

[lasvay's  reagent  i-  prepared  as  follows:  0.5  gramme  of  sulph- 
anilic  acid  in  150  c.c.  of  dilute  acetic  acid  i-  treated  with  0.1 
gramme  of  naphtylamin,  and  dissolved  in  20  c.c.  of  boiling  water. 


114  THE  DIGESTIVE  FLUIDS. 

After  standing  for  some  time  the  supernatant  fluid  is  poured  off,  and 
the  blue  sediment  dissolved  in  150  c.c.  of  dilute  acetic  acid.  The 
solution  is  kept  in  a  sealed  bottle. 

Extractives. — Of  extractives,  normal  saliva  contains  a  small 
amount  of  urea,  and  traces  of  cholesterin,  lecithin,  and  leucin.  In 
gouty  conditions  uric  acid  has  been  found  ;  sugar,  the  biliary  pig- 
ments, and  biliary  acids  are  not  eliminated  through  the  saliva. 

Mineral  Constituents. — The  mineral  constituents  of  saliva  con- 
sist to  the  extent  of  90  to  92  per  cent,  of  soluble  salts,  among  which 
the  chlorides  greatly  predominate,  and  of  about  6  per  cent,  of  salts, 
which  are  principally  represented  by  the  carbonates  and  phosphates 
of  calcium  and  magnesium,  which  are  held  in  solution  by  the  free 
carbonic  acid  of  the  saliva.  In  addition,  a  trace  of  iron  is  found. 
Following  the  administration  of  bromides  and  iodides  a  notable 
elimination  of  these  salts  occurs  through  this  channel. 

Gases. — Of  gases,  which  are  present  in  a  state  of  solution,  we 
find  about  20  c.c.  for  every  100  grammes  of  saliva.  Of  these,  19 
c.c.  are  represented  by  carbon  dioxide,  while  oxygen  and  nitrogen 
together  amount  to  only  1  c.c. 

THE  GASTRIC  JUICE. 

General  Considerations. — The  gastric  juice  is  the  secretory 
product  of  the  glandular  structures  of  the  stomach,  and  the  only 
digestive  fluid  which  presents  an  acid  reaction.  In  pure  form  it  is 
best  obtained  from  animals  after  ligating  the  ducts  of  the  salivary 
glands  and  establishing  a  fistulous  opening  on  the  outer  abdominal 
walls.  If  the  mucous  membrane  is  then  appropriately  stimulated, 
a  clear  or  but  slightly  opalescent  yellowish  fluid  is  obtained,  which 
has  a  very  characteristic  odor  and  a  strongly  acid  reaction.  Its 
density  varies  between  1.001  and  1.010. 

On  microscopic  examination  are  found  epithelial  cells  from  the 
lining  of  the  glandular  ducts,  goblet-cells,  mucous  corpuscles,  free 
nuclei,  and  a  variable  number  of  bacteria.  In  addition,  we  often 
observe  small  tapioca-like  bodies,  which  under  the  microscope  are 
seen  to  contain  numerous  formations  resembling  snail-shells,  and 
which  probably  consist  of  altered  mucin. 

Amount. — Of  the  total  amount  of  gastric  juice  secreted  in  the 
twenty-four  hours,  but  little  is  known.  Its  secretion  is  influenced  by 
numerous  factors,  such  as  the  appetite,  the  quality  and  quantity  of 
the  food  ingested,  the  age  and  sex  of  the  individual,  the  time  of  day 
(notably  in  relation  to  the  taking  of  food),  the  various  emotions,  etc. 
According  to  Bidder  and  Schmidt,  the  amount  corresponds  to  about 
one-tenth  of  the  body-weight,  so  that  a  man  weighing  70  kilo- 
grammes would  secrete  about  7000  grammes  in  the  twenty-four 
hours.  This  figure,  however,  I  regard  as  too  high,  and  am  inclined 
to  place  the  amount  at  from  2000  to  3000  c.c. 

The  non-digesting  stomach  of  the  dog  and  other  animals  is  said 


THE  GASTRIC  JUICE.  115 

to  contain  no  fluid  ;  in  man,  however,  a  small  amount  of  gastric 
juice  can  usually  be  obtained  by  means  of  the  stomach-tube,  vary- 
ing between  1  and  60  c.c.  Larger  amounts  may  be  found  under 
pathologic  conditions,  and  in  the  so-called  Magensaftfluss  of  the 
Germans  it  is  not  rare  to  find  as  much  as  1000  c.c.  in  the  early 
morning,  before  any  food  has  been  taken. 

The  amount  of  fluid  which  can  be  normally  obtained  from  the 
digesting  organ  is  likewise  variable.  It  depends  upon  the  amount 
of  liquid  ingested,  the  period  of  digestion,  the  character  of  the  food, 
the  size  and  motor  power  of  the  stomach,  etc.  Exact  figures,  how- 
ever, are  lacking  to  represent  these  relations,  and  it  is  manifest  that 
such  figures  must  always  have  reference  to  more  or  less  diluted 
gastric  juice. 

Chemical  Composition. — A  general  idea  of  the  chemical  com- 
position of  the  gastric  juice  may  be  formed  from  the  following  analy- 
ses, which  are  taken  from  C.  Schmidt  ;  but  it  is  to  be  noted  that 
the  specimen  of  human  gastric  juice  was  contaminated  with  saliva 
and  somewhat  diluted  with  water  (the  figures  have  reference  to  1000 
parts) : 

Human  gastric  juice,  Gastric  juice  of 

containing  saliva  dog,  free 

with   water.  from  saliva. 

Water 994.40 973.0 

Solids      5.60 27.0 

Organic  material 3.10 17.1 

Mineral  salts 2.19 6.7 

Sodium  chloride 1.46 2.5 

Calcium  chloride      0.06 0.6 

Potassium  chloride 0.55  .......  1.1 

Ammonium  chloride 0.5 

Calcium  phosphate 1.7 

Magnesium  phosphate 0.2 

Iron 0.12 0.1 

Free  hydrochloric  acid 0.20 0.1 

Acidity  of  the  Gastric  Juice. — It  has  now  been  definitely 
established  that  the  acidity  of  normal  gastric  juice  is  referable  to 
the  presence  of  free  hydrochloric  acid,  and  to  this  only.  This  can 
be  shown  by  estimating  the  amount  of  chlorine  and  all  basic  sub- 
stances that  are  present,  when  it  will  bo  found  that  after  the  acid 
affinities  of  tin-  latter  have  been  saturated,  a  certain  amount  of 
chlorine  still  remains,  which  can  be  referable  only  to  hydrochloric 
acid,  and  corresponds  in  its  degree  of  acidity  to  that  observed  in 
th<-    gastric    juice    itself. 

During  the  process  of  digestion,  however,  other  factors  enter  into 
consideration.      In   the  beginning  of  digestion   lactic  acid  is  always 

{tre-eiit  when  carbohydrates  form  part  of  the  meal.  Its  amount, 
lowever,  i-  then  quite  small,  and  after  the  ingestion  of  Ewald's 
test-breakfkst,  for  example,  does  not  exceed  0.3  pro  niille.  The 
occurrence  of  larger  quantities  of  lactic  acid,  as  from  1  to  .">  pro 
mille,  is  always  abnormal,  and  in  many  cases  indicative  of  the 
existence    of   carcinoma   of    the  stomach.      During  the   later  stages 


116  THE  DIGESTIVE  FLUIDS. 

of  digestion,  when  hydrochloric  acid  is  found  in  a  free  state,  lactic 
acid  disappears.  Its  origin,  under  normal  conditions  at  least,  is 
referable  to  the  action  of  certain  bacteria,  such  as  the  Bacterium 
lactis,  the  Bacillus  lactis  aerogenes,  etc.,  upon  starches  and  sugars, 
as  represented  by  the  equations  : 

(1)  2C5HI0O5  +  H20  =  C12H22On 

Starch.  Lactose. 

(2)  C12H22CU  +  H20  =  4C,H603 

Lactose.  Lactic  acid. 

Other  organic  acids,  such  as  butyric  acid  and  acetic  acid,  are  usu- 
ally not  found  in  the  gastric  contents  unless  large  amounts  of  milk, 
carbohydrates,  or  alcohol  have  been  ingested.  In  such  event,  how- 
ever, they  may  be  present,  and,  like  lactic  acid,  are  then  referable  to 
the  action  of  certain  micro-organisms.  They  are  essentially  of  patho- 
logical significance. 

It  is  thus  seen  that  even  during  the  process  of  digestion  the 
acidity  of  the  gastric  contents  is,  under  normal  conditions,  scarcely 
influenced  by  acids  other  than  hydrochloric  acid.  It  should 
be  noted,  however,  that  following  the  ingestion  of  food  hydro- 
chloric acid  does  not  appear  in  a  free  state  at  once,  but  only 
after  the  affinities  of  the  albuminous  constituents  of  the  food  have 
been  saturated.  We  consequently  find  that  while  in  the  begin- 
ning of  digestion  the  acidity  of  the  stomach-contents  is  largely 
referable  to  such  combined  acid,  in  the  later  phases  of  digestion  two 
factors  enter  into  consideration,  viz.,  free  and  combined  hydrochloric 
acid.  The  period  at  which  free  acid  appears  as  such  varies,  of 
course,  with  the  character  of  the  meal,  and  directly  with  the  amount 
of  proteids  ingested.  After  the  administration  of  Ewald's  test- 
breakfast  free  acid  is  thus  found  only  at  the  expiration  of  about 
thirty-five  minutes  ;  while  after  the  administration  of  Riegel's  test- 
dinner,  which  contains  much  larger  amounts  of  albumin,  two  hours 
must  elapse  before  free  acid  can  be  demonstrated. 

Acid  salts,  finally,  play  only  a  small  part  in  determining  the  total 
acidity  of  the  gastric  juice  ;  and  it  is  thus  clear  that  unless  carbo- 
hydrates, much  fat,  and  alcohol  have  been  ingested,  hydrochloric 
acid,  either  in  a  free  state  or  in  combination  with  albumin,  or  both, 
is  the  sole  factor  which  enters  into  consideration.  Under  pathological 
conditions,  on  the  other  hand,  lactic  acid,  butyric  acid,  and  acetic 
acid  may  also  play  a  part ;  but  then  hydrochloric  acid  is  usually  not 
present,  and  the  acidity  of  the  gastric  contents  is  hence  largely 
referable  to  fermentative  changes  which  have  taken  place  in  the 
stomach. 

Determination  of  the  Total  Acidity  of  the  Gastric  Contents. 
— Five  or  10  c.c.  of  the  filtered  gastric  contents  are  titrated  with  a 
decinormal  solution  of  sodium  hydrate,  using  phenolphthalein  as  an 
indicator,  until  the  rose  color,  which  appears  on  the  addition  of  each 
drop  of  the  sodium  hydrate  solution,  no  longer  disappears  on  stirring 
or  is  intensified  by  the  addition  of  a  further  drop.     The  number  of 


THE  GASTRIC  JUICE.  117 

cubic  centimeters  employed  to  bring  about  this  reaction,  multiplied 
by  0.00365,  indicates  the  acidity  of  the  5  or  10  c.c.  of  gastric  juice 
in  terms  of  hydrochloric  acid. 

Amount. — The  degree  of  acidity  of  the  gastric  juice  is  usually 
fairly  constant,  and  in  man  varies  between  0.15  and  0.25  per  cent. 
It  is  influenced  to  a  certain  extent  by  the  character  of  the  food ;  for 
instance,  following  the  administration  of  a  meal  rich  in  proteids, 
somewhat  larger  amounts  are  obtained  than  after  the  ingestion  of 
carbohydrates  or  fats.  The  smallest  amounts  are  found  after  the 
ingestion  of  water.  After  Ewald's  test-breakfast,  which  consists  of 
from  35  to  70  grammes  of  wheat  bread  and  300  to  400  c.c.  of 
water,  or  weak  tea  without  sugar,  the  maximum  acidity  is  reached 
in  about  one  hour,  and  corresponds  to  1.5  to  1.75  pro  mille.  Follow- 
ing the  ingestion  of  Riegel's  test-meal,  on  the  other  hand,  which 
eon-ists  of  a  plate  of  soup  (400  c.c),  200  grammes  of  beefsteak,  50 
gramme-;  of  wheat  bread,  and  200  c.c.  of  water,  the  amount  of 
hydrochloric  acid  increases  to  2.7  pro  mille,  after  from  one  hundred 
and  eighty  to  two  hundred  and  ten  minutes.  In  disease  still  higher 
figures  (5  p.  m.)  may  be  observed  ;  or  its  secretion  may  diminish 
below  the  normal,  and  may  even  cease  altogether. 

Hydrochloric  Acid. — Origin. — The  hydrochloric  acid  of  the 
gastric  juice  is  furnished  by  the  so-called  parietal,  adelomorphous,  or 
oxyntic  cells,  which  are  principally  found  in  the  glands  of  the 
fundus.  This  can  be  demonstrated  by  resecting  the  fundus,  then 
closing  one  end  with  a  fine  suture,  and  sewing  the  other  into  the 
abdominal  wound,  while  the  cardiac  portion  of  the  stomach  is 
joined  to  the  pyloric  end.  If  food  be  now  administered  to  the 
animal,  a  fluid  will  be  secreted  by  the  isolated  fundus  in  which  the 
presence  of  free  hydrochloric  acid  can  easily  be  shown.  If,  on  the 
other  hand,  the  pyloric-  end  of  the  stomach,  in  which  no  parietal 
cells  arc  found,  is  similarly  isolated,  no  acid  is  obtained,  but,  instead, 
a  Btrongly  alkaline  mucus. 

While  it  is  thus  clear  that  the  hydrochloric  acid  is  furnished  by 
the  parietal  eel!-,  we  are  as  yet  ignorant  of  the  mechanism  by  which 
this  is  accomplished.  A  free  acid  is  manifestly  not  present  in  these 
cells,  as  can  be  shown  by  testing  with  litmus-paper,  or  still  better  by. 
injecting  potassium  ferrocyanide  and  lactate  of  iron  into  the  circula- 
tion of  an  animal,  when  it  will  be  observed  that  JJerlin-blue  is 
formed  in  the  Stomach-cavity,  while  the  cells  themselves  remain 
unstained,  it  thus  follows  that  a  substance  must  either  be  present 
in  tin-  cells  which  is  capable  of  yielding  hydrochloric  acid  when 
secreted  to  the  outside,  or  n  mechanism  must  exist  by  which  the 
hydrochloric  acid,  though  formed  within  the  cells,  is  at  once  elim- 
inated. The  hitter  v;ew  i<  now  generally  held.  That  the  hydro- 
chloric acid  i-  derived  from  the  chlorides  of  the  blood  can  be 
regarded  ;i-  an  established  feet.  It  may  thus  be  secreted  even 
though  no  food— tuff-  have  been  ingested;  and  Kahn,  moreover,has 

shown  that    animals  in  which    the  chlorides   of  the    bodv  have    been 


118  THE  DIGESTIVE  FLUIDS. 

artificially  reduced  to  that  minimum  which  is  tenaciously  retained 
are  no  longer  capable  of  secreting  hydrochloric  acid.  The  mech- 
anism, however,  by  which  the  formation  of  hydrochloric  acid  takes 
place  is,  as  has  been  stated,  unknown.  Ludwig  formerly  thought 
that  it  resulted  through  electrolytic  influences  within  the  cells,  and 
that  the  acid  then  diffused  out  into  the  lumen  of  the  glandular  duct 
before  the  alkaline  elements  of  the  cell  could  bring  about  its  neutral- 
ization. This,  however,  is  improbable,  and  the  view  that  is  now 
held  by  most  observers  is  that  expressed  by  Maly,  according  to 
which  the  hydrochloric  acid  results  from  a  mass-action  on  the  part 
of  the  carbonic  acid  of  the  blood  upon  the  chlorides  in  the  body  of 
cells,  and  is  immediately  eliminated  to  the  outside,  while  the  result- 
ing bicarbonate  is  returned  to  the  blood.  We  know  that  within  the 
cells  carbon  dioxide  is  present  under  great  pressure.  Schlierbeck 
thus  found  that  water  which  is  introduced  into  the  stomach  of  living 
dogs  after  a  variable  length  of  time  contains  a  certain  amount  of 
carbon  dioxide,  and  that  its  tension  rises  from  30  to  40  Hgram. 
while  fasting,  to  130  to  140  Hgmm.  during  the  process  of  active 
digestion. 

Of  late,  Liebermann  has  further  suggested  that  lecithalbumin  may 
be  present  in  the  parietal  cells  in  combination  with  sodium  chloride, 
and  that  hydrochloric  acid  may  result  from  this  through  a  mass- 
action  on  the  part  of  carbonic  acid,  which,  in  turn,  is  formed  within 
the  cells  as  a  result  of  an  increase  of  their  functional  activity. 

Significance  of  the  Hydrochloric  Acid. — The  assumption  that  the 
principal  function  of  the  hydrochloric  acid  consists  in  its  power  to 
render  the  pepsin  of  the  gastric  juice  physiologically  active,  viz., 
capable  of  bringing  about  the  transformation  of  albumins  into  albu- 
moses  and  peptones,  has  now  been  largely  abandoned.  We  know 
that  life  can  go  on  in  the  entire  absence  of  the  stomach,  as  has  been 
proved  not  only  by  experiments  on  animals,  but  also  by  operations 
which  have  been  performed  on  the  human  being.  A  dog  whose 
stomach  was  almost  entirely  removed  by  Czerny  in  1876,  lived  for 
more  than  six  years  after  the  operation,  when  it  was  killed  in  Lud- 
wig's  laboratory ;  and  it  is  reported  that  the  animal  was  normal  in 
every  respect,  and  had  increased  in  weight  from  5850  grammes  to 
7000  grammes.  It  is  thus  manifest  that  while  the  hydrochloric  acid 
of  the  gastric  juice  no  doubt  aids  in  the  process  of  albuminous  di- 
gestion, its  presence  to  this  end  is  not  imperative,  and  the  question 
naturally  suggests  itself,  whether  the  secretion  of  such  large  amounts 
of  acid  does  not  serve  another  and  perhaps  more  important  purpose. 
This  purpose  is  now  thought  to  be  the  prevention  of  putrefactive 
changes  in  the  contents  of  the  stomach,  and  we  find,  as  a  matter  of 
fact,  that  albuminous  material  that  has  been  removed  from  the 
stomach  at  the  height  of  digestion  can  be  preserved  for  a  long  time 
without  undergoing  decomposition.  It  has  been  noted,  moreover, 
that  the  gastric  juice  is  also  capable  of  arresting  putrefactive  proc- 
esses when  these  have  begun  before  the  ingestion  of  such  materiaL 


THE   GASTRIC  JUICE.  119 

This  problem  has  been  carefully  investigated,  and  we  now  know 
that  the  amount  of  hydrochloric  acid  which  is  present  at  the  height 
of  digestion  is  sufficient  at  least  to  arrest  the  activity  of  most 
bacteria  which  are  usually  ingested  together  with  food.  Some  of 
these,  however,  are  more  resistant  than  others,  and  among  them  the 
lactic  acid  bacteria  are  especially  stable.  We  can  accordingly  under- 
stand why  in  the  beginning  of  the  process  of  digestion  traces  of 
lactic  acid  are  usually  found.  So  soon,  however,  as  the  percen- 
tage of  hydrochloric  acid  has  increased  to  0.7  pro  mille  the  activity 
of  these  organisms  also  ceases.  Whether  or  not  the  gastric  jnice 
actually  destroys  all  of  the  more  common  micro-organisms  that 
are  swallowed  has  not  been  determined,  but  there  is  evidence  to 
show  that  in  some  instances  at  least  the  spores  remain  unaffected 
and  can  later  develop  in  the  alkaline  contents  of  the  small  intes- 
tine. In  this  manner  we  can  account  for  the  presence  of  the 
innumerable  bacteria  which  are  found  in  the  lower  intestinal 
tract.  It  must  be  remembered,  moreover,  that  in  the  beginning 
of  digestion — i.  r.}  when  free  hydrochloric  acid  is  as  yet  not  pres- 
ent— large  numbers  of  bacteria  may  also  pass  through  the  stomach 
unaffected,  as  combined  hydrochloric  acid  possesses  no  anti-fermenta- 
tive properties.  Some  of  the  more  important  pathogenic  bacteria 
are  unfortunately  more  resistant  than  the  common  benign  forms. 
This  is  true  especially  of  the  tubercle  bacillus,  and  in  many  cases  of 
the  anthrax  bacillus  and  some  of  the  pus  organisms,  while  the 
cholera  bacillus,  on  the  other  hand,  is  readily  destroyed. 

Tests  for  Free  Hydrochloric  Acid. — A  large  number  of  tests  have 
been  devised  for  the  purpose  of  demonstrating  the  presence  of  free 
hydrochloric  acid  in  the  stomach-contents.  At  this  place  only  the 
more  important  ones  will  be  described,  which  are  employed  in  the 
clinical   laboratory. 

Topper's  Test. — A  small  amount  of  the  filtered  gastric  contents 
is  treated  with  a  few  drops  of  a  0.5  per  cent,  alcoholic  solution  of 
dimethyl-amido-azobenzol,  when  in  the  presence  of  free  hydro- 
chloric acid  a  beautiful  cherry-red  color  develops  at  once,  which 
varies  in  intensity  with  the  amount  of  the  free  acid  present.  Com- 
bined hydrochloric  acid,  as  well  as  acid  salts  and  organic  acids,  in 
the  concentration  in  which  they  may  be  met  with  in  the  stomach- 
conterits,  does  not  produce  this  color. 

The  delicacy  of  the  reagent  is  such  that  the  norma]  yellow  color 
of  the  indicator  is  changed  to  a  reddish  tinge  upon  the  addition  of 
hut  one  drop  of  a  X  normal  solution  of  hydrochloric  acid  in  5  cc 
of  distilled  water,  viz.,  0.7  per  cent. 

GtTNZBURo/s  Test. — The  reageni  consists  of  2  grammes  of  phloro- 
glucin  and  1  gramme  of  vanillin,  dissolved  in  100  grammes  of 
80  per  cent,  alcohol.  It  should  be  kept  in  a  dark-colored, glass- 
stoppered  bottle. 

A  few  drops  of  the  filtered  gastric  content-  are  carefully  evapo- 
rated with  an  equal  amount  of  the  reageni  on  a  plate  of  thin  porce- 


120  THE  DIGESTIVE  FLUIDS. 

lain  or  glass,  when  in  the  presence  of  free  hydrochloric  acid  a 
rose-colored  mirror  is  obtained,  which  varies  in  intensity  with  the 
amount  of  the  acid. 

Organic  acids  do  not  produce  the  reaction. 

The  delicacy  of  the  test  is  such  that  the  presence  of  0.05  gramme 
of  hydrochloric  acid  in  100  parts  of  water  can  be  demonstrated. 

Boas'  Test. — The  reagent  consists  of  5  grammes  of  resublimed 
resorcin  and  3  grammes  of  cane-sugar,  dissolved  in  100  grammes 
of  94  per  cent,  alcohol.  The  test  is  conducted  like  that  of  Giinz- 
burg,  but  it  is  necessary  to  heat  a  little  more  strongly,  especially 
after  the  fluid  has  been  evaporated.  A  similarly  colored  mirror  is 
obtained,  which  gradually  fades  on  cooling. 

The  delicacy  of  the  test  is  the  same  as  that  of  Giinzburg. 

Examination  for  the  Presence  of  Combined  Hydrochloric  Acid. — 
The  presence  of  combined  hydrochloric  acid  cannot  be  demonstrated 
by  means  of  simple  tests  like  those  just  described,  but  is  inferred 
indirectly,  as  shown  in  the  following  method  : 

Separate  Estimation  of  the  Free  and  Combined  Hydrochloric  Acid 
of  the  Gastric  Contents. — Topfer's  Method. — The  total  acidity  of 
a  given  amount  of  the  gastric  contents  is  first  determined,  as 
already  described,  and  termed  A.  This  indicates  the  amount  of 
the  physiologically  active  hydrochloric  acid,  viz.,  the  free  and  the 
combined  hydrochloric  acid,  as  well  as  that  of  any  acid  salts  and 
organic  acids  that  may  be  present. 

In  a  second  specimen  the  total  amount  of  free  acids  and  acid 
salts  is  determined  by  titrating,  as  before,  with  a  -^  normal  solu- 
tion of  sodium  hydrate,  but  using  a  few  drops  of  a  1  per  cent, 
aqueous  solution  of  alizarin  (alizarin-monosulphonate  of  sodium)  as 
an  indicator.  The  titration  is  carried  to  a  point  Mrhere  a  pure  violet 
color  is  obtained.  The  result  is  termed  B.  The  difference  between 
A  and  B  is  thus  referable  to  the  presence  of  the  combined  hydro- 
chloric acid,  and  termed  C. 

In  a  third  specimen  the  amount  of  free  hydrochloric  acid  is 
determined  by  titrating  with  the  decinormal  solution  of  sodium 
hydrate,  using  a  few  drops  of  a  0.5  per  cent,  alcoholic  solution  of 
dimethyl-amido-azobenzol  as  indicator,  until  the  red  color  which 
first  appears  has  changed  to  yellow.  The  result  is  termed  F. 
F  plus  C  will  then  represent  the  amount  of  physiologically  active 
hydrochloric  acid  P,  viz.,  the  combined  and  free  acid,  while  the 
difference  between  A  and  P  corresponds  to  the  acid  salts  and 
organic  acids  that  may  also  be  present. 

Method  of  Morner  and  Sjoqvist. — By  this  method  the  amount 
of  physiologically  active  hydrochloric  acid  can  also  be  estimated.  It 
is  somewhat  more  complicated  and  time-consuming  than  the  one  just 
described,  but  more  accurate.  It  is  based  upon  the  fact  that  on 
evaporating  the  gastric  contents  to  dryness  in  the  presence  of  barium 
carbonate,  and  subsequently   incinerating  the    residue,  the  organic 


THE  GASTRIC  JUICE.  121 

acids  are  destroyed,  while  the  hydrochloric  acid  combines  with  the 
barium,  and  can  thus  be  estimated  as  barium  chloride. 

To  this  end,  10  c.c.  of  the  filtered  stomach-contents  are  treated 
with  a  pinch  of  chemically  pure  barium  carbonate  and  evaporated  to 
dryness.  The  residue  is  ignited  at  a  moderate  temperature  until 
white,  and  the  remaining  ash  extracted  with  hot  water.  After 
filtering  the  solution  (about  50  c.c.)  it  is  treated  with  an  equal  volume 
of  alcohol  (94  per  cent.)  and  0.75  c.c.  of  a  10  per  cent,  solution  of 
sodium  acetate  in  dilute  acetic  acid  (10  per  cent,  solution).  The 
barium  is  then  estimated  by  titrating  with  a  standardized  solution  of 
potassium  bichromate,  containing  8.5  grammes  of  the  chemically 
pure  substance  in  the  liter,  and  using  tetramethyl-paraphenyldiamin 
paper  as  an  indicator,  until  a  drop  of  the  titrated  fluid  causes  a  distinct 
blue  coloration  of  the  paper  within  one  minute.  From  the  number 
of  cubic  centimeters  employed  to  bring  about  the  end-reaction  the 
corresponding  amount  of  hydrochloric  acid  can  then  be  calculated 
by  multiplying  with  0.00405,  if  the  bichromate  solution  was 
standardized  with  a   ^  normal  solution  of  barium  chloride. 

Method  of  Leo. — This  method  is  based  upon  the  observation 
that  calcium  carbonate  combines  with  free  and  loosely  bound  hydro- 
chloric acid  at  ordinary  temperatures  to  form  neutral  calcium  chlo- 
ride, while  the  acid  phosphates  are  not  affected.  If  then  the  total 
acidity  of  the  stomach-contents  is  first  determined,  and  the  acidity 
referable  to  acid  salts  deducted  from  this  figure,  the  amount  of 
physiologically  active  hydrochloric  acid  is  ascertained.  Organic 
acids,  of  course,  must  first  be  removed  by  extracting  with  ether 
(50-100  c.c.  for  10  c.c.  of  gastric  juice).  As  the  monophosphates  of 
potassium  and  sodium,  however,  are  changed  to  monocalcium  phos- 
phate in  the  presence  of  calcium  chloride,  which  requires  double  the 
quantity  of  sodium  hydrate  solution  for  its  neutralization  than  the 
corresponding  amount  of  the  alkaline  phosphates,  it  is  either  neces- 
sary to  divide  the  number  of  cubic  centimeters  of  the  sodium 
hydrate  solution  which  is  used  in  the  second  titration  by  2,  or  to 
make  the  firsl  titration  under  the  same  conditions  as  the  first,  viz., 
after   adding   an   excess  of  calcium  chloride  solution. 

To  this  end,  then,  we  proceed  as  follows:  15  c.c.  of  the  filtered 
gastric  contents  are  treated  with  a  pinch  of  dry  and  chemically 
pure  calcium  carbonate.  The  mixture  is  well  stirred  and  passed  at 
once  through  a  dry  filter,  'fen  c.c,  from  which  the  carbon  dioxide 
is  expelled  by  a  current  of  air,  are  then  treated  with  5  cv.  of  a  con- 
centrated solution  of  calcium  chloride  and  titrated  as  usual.  The 
resulting  value  is  termed  I',  and  represents  the  acid  phosphates. 
The  total  acidity  is  then  determined  in  another  specimen,  after 
adding  the  same  amounl  of  the  calcium  chloride  solution,  and  the 
result  termed  T.  T  minus  A  will  then  represent  the  amount  of  the 
physiologically   active   hydrochloric  acid. 

The  combined  hydrochloric  acid  may,  of  course,  he  readily  deter- 
mined    with     either    of    the    two    method-    which     have    jnst     been 


122  THE  DIGESTIVE  FLUIDS. 

described,  by  separately  estimating  the  amount  of  free  hydrochloric- 
acid  by  Topfer's  method,  and  deducting  the  result  from  the  total 
amount  of  the  physiologically  active  acid.  More  accurate  results 
are  probably  reached  in  this  manner  than  with  Topfer's  method, 
unless  some  experience  has  been  gained  in  the  titration  with  alizarin. 

Should  organic  acids  also  be  present,  their  amount  may  be  esti- 
mated by  deducting  from  the  total  acidity  the  result  reached  with 
the  above  method. 

If  monophosphates  are  present  at  the  same  time,  the  resulting 
figures  will  be  a  little  too  low ;  but  the  error  which  is  thus  incurred 
is  trifling.  It  may  be  obviated,  however,  by  making  use  of  Leo's 
method  (see  above). 

Lactic  Acid. — Tests  for  Lactic  Acid. — In  order  to  assure  our- 
selves that  any  lactic  acid  that  may  be  found  in  the  gastric  contents 
has  not  been  introduced  into  the  stomach  from  without,  it  is  neces- 
sary to  make  such  examinations  after  the  administration  of  a  test- 
meal,  in  which  the  acid  in  question  does  not  occur  preformed.  The 
meal  which  is  almost  exclusively  used  for  this  purpose  in  clinical 
work  is  the  so-called  test-meal  of  Boas.  It  consists  of  a  plateful 
of  oatmeal  soup,  which  is  prepared  by  adding  a  tablespoonful  of 
rolled  oats  and  a  little  salt  to  a  liter  of  water,  and  boiling  down  to 
about  500  c.c.  The  contents  of  the  stomach  are  then  drawn  off 
after  one  hour,  filtered,  and  treated  as  described  below. 

Uffelmann's  Test. — About  10  c.c.  of  the  filtered  gastric  con- 
tents are  extracted  with  ether  (50-100  c.c.)  by  shakiug  in  a  sepa- 
rating funnel  for  from  twenty  to  thirty  minutes.  The  ethereal  ex- 
tract is  then  evaporated  to  dryness  by  distilling  on  a  water-bath  ; 
the  residue  is  taken  up  with  a  few  cubic  centimeters  of  distilled 
water,  and  treated  as  follows :  3  drops  of  a  saturated  aqueous  solu- 
tion of  ferric  chloride  are  mixed  with  an  equal  number  of  drops  of  a 
concentrated  solution  of  pure  carbolic  acid,  and  diluted  with  water 
until  a  light-amethyst  color  is  obtained.  To  this  solution  a  portion 
of  the  ethereal  extract  is  added,  when  in  the  presence  of  lactic  acid  a 
lemon  or  canary  color  develops. 

The  delicacy  of  the  test  is  such  that  the  presence  of  0.1  per  cent, 
of  lactic  acid  can  be  demonstrated. 

Kelling's  Test. — Five  or  10  c.c.  of  the  filtered  stomach-contents 
are  diluted  from  ten  to  twenty  times  with  water  and  treated  with  1 
or  2  drops  of  a  5  per  cent,  aqueous  solution  of  ferric  chloride.  In 
the  presence  of  lactic  acid  a  distinct  greenish-yellow  color  is  obtained 
when  the  tube  is  held  to  the  light. 

Boas'  Test. — This  test  is  more  accurate  than  the  two  just  de- 
scribed, but  more  time-consuming  and  complicated.  It  is  based 
upon  the  decomposition  of  lactic  acid  into  formic  acid  and  acetic 
aldehyde,  and  the  demonstration  of  the  presence  of  the  latter.  To 
this  end,  from  10  to  20  c.c.  of  the  filtered  stomach-contents  are- 
treated  with  a  slight  excess  of  barium  carbonate,  and  evaporated  on 
a   water-bath.     The  resulting  syrup  is  acidified  with  a  few  drops 


THE  GASTRIC  JUICE.  123 

of  phosphoric  acid,  and  freed  from  carbon  dioxide  by  momentary 
ebullition.  On  cooling,  it  is  extracted  with  100  c.c.  of  ether  by 
shaking  for  about  thirty  minutes.  The  ethereal  extract  is  poured 
off,  the  ether  distilled,  and  the  residue  taken  up  with  45  c.c.  of 
water.  After  filtering,  the  solution  is  then  treated  with  5  c.c.  of 
concentrated  sulphuric  acid  and  a  pinch  of  manganese  dioxide,  and 
carefully  heated  to  boiling.  Should  lactic  acid  be  present,  this  is 
now  decomposed,  and  acetic  aldehyde  liberated,  which  ran  be  demon- 
strated by  passing  the  vapor  into  a  test-tube  containing  Nessler's 
reagent  or  an  alkaline  solution  of  iodopotassic  iodide.  In  the  first 
instance,  yellowish-red  aldehyde  of  mercury  is  formed,  while  iodoform 
results  in  the  latter,  and  can  be  readily  recognized  from  its  odor, 
which  becomes  marked  when  the  solution  is  heated. 

Tests  for  Acetic  Acid  and  Butyric  Acid. — These  acids  can 
usually  be  recognized  by  their  odor.  Chemically  they  can  be 
demonstrated  as  follows  : 

Test  for  Acetic  Acid. — Ten  c.c.  of  the  filtered  stomach-contents 
are  extracted  with  ether  as  above.  The  ether  is  distilled  off,  the 
residue  taken  up  with  a  few  drops  of  water  and  accurately  neutral- 
ized with  sodium  hydrate.  To  this  solution  a  drop  or  two  of  a  very 
dilute  solution  of  ferric  chloride  is  added,  when  in  the  presence  of 
acetic  acid  a  dark-red  color  develops.  With  nitrate  of  silver,  on  the 
other  hand,  a  precipitate  is  obtained  which  is  soluble  in  hot  water. 

Test  for  Butyric  Acid. — The  ethereal  extract  of  10  c.c.  of  the 
stomach-contents  is  freed  from  ether  by  distillation,  the  residue  is 
dissolved  in  a  few  cubic  centimeters  of  water,  and  treated  with  a  trace 
of  calcium  chloride  in  substance.  In  the  presence  of  butyric  acid 
small  oil  droplets  separate  out,  the  nature  of  which  is  readily  recog- 
nized from  the  pungent  odor.  If,  in  the  place  of  calcium  chloride, 
a  slight  excess  of  baryta-water  is  used,  highly  refractive  rhombic 
platelets  or  granular,  wart-like  masses  are  obtained  on  evaporation, 
which  consist  of  barium   butyrate. 

Butyric  acid  can  also  be  recognized  by  the  peculiar  odor  of  pine- 
apple which  develops  when  the  dry  residue  of  the  ethereal  solution 
is  treatfd  with  a  little  sulphuric  acid  and  alcohol.  The  reaction  is 
due  to  the  formation  of  butyl  ethylate,  C,H702.C2H5. 

Quantitative  Estimation  of  Lactic  Acid. — This  is  best  accom- 
plished by  means  of  Boat?  method:  The  decomposition  of  the  lactic 
acid  is  effected  as  described  above.  After  the  addition  of  the  sul- 
phuric add  and  manganese  dioxide  the  flask  is  closed  with  a  doubly 
perforated  stopper.  Through  one  aperture  a  bent  lube  passes  to  the 
condenser,  while  a  Btraighl  tube  passes  through  the  other  opening, 
and  i-  provided  at  its  i'v<-<-  end  with  a  small  piece  of  rubber  tubing 
that  is  clamped  ;  thi~  tube  should  dip  well  into  the  liquid,  and  serves 
for  passing  a  current  of  air  through  the  solution  when  the  distilla- 
tion i-  completed.      The   mixture   is   then   distilled    until  about  four- 

lif'th-  of  the  contents  have  passed  over,  excessive  heat  being  carefully 

.avoided,  -(,  :i-  to  prevent    decomposition  of  the   aldehyde-.      The  dis- 


124  THE  DIGESTIVE  FLUIDS. 

tillate,  which  is  received  in  a  high  Erlenmeyer  flask,  is  heated  with 
20  c.c.  of  a  Jq-  normal  solution  of  iodine  and  the  same  amount  of  a 
5.6  per  cent,  solution  of  potassium  hydrate.  The  mixture  is  thor- 
oughly shaken  and  set  aside  for  a  few  minutes.  The  excess  of 
iodine  is  then  estimated  after  adding  20  c.c.  of  hydrochloric  acid 
(specific  gravity  1.018),  by  titrating  with  a  -^  normal  solution  of 
sodium  thiosulphate.  The  titration  is  carried  almost  to  the  point  of 
decolorization,  when  a  little  starch  solution  is  added,  and  the  titra- 
tion continued  until  the  last  trace  of  blue  has  disappeared.  The  dif- 
ference between  the  number  of  cubic  centimeters  of  the  thiosulphate 
solution  employed  to  bring  about  this  end  and  the  amount  of  the  iodine 
solution  added,  viz.,  20,  will  indicate  the  number  of  cubic  centimeters 
of  the  latter  which  were  utilized  in  the  formation  of  iodoform.  By 
multiplying  this  number  by  0.003388  the  corresponding  amount 
of  lactic  acid  is  ascertained. 

Quantitative  Estimation  of  the  Organic  Acids. — The  organic  acids 
in  toto  may  be  estimated  by  one  of  the  methods  already  described 
(pages  120  and  121),  or  according  to  the  following  procedure,  as 
suggested  by  Hehner-Seemann.  This  method  is  based  upon  the 
transformation  of  the  organic  acids  into  their  alkaline  salts,  and 
their  subsequent  combustion,  with  the  formation  of  the  correspond- 
ing carbonates,  which  are  then  estimated  by  titration.  At  the  same 
time  the  amount  of  physiologically  active  hydrochloric  acid  can  be 
obtained  as  follows  :  10  c.c.  of  the  filtered  gastric  contents  are  neu- 
tralized with  a  decinormal  solution  of  sodium  hydrate,  and  the  total 
acidity  thus  ascertained.  The  neutralized  solution  is  then  evaporated 
to  dryness  and  the  residue  incinerated.  Care  should  be  had,  however, 
that  the  application  of  heat  is  discontinued  as  soon  as  the  ash  has 
ceased  to  burn  with  a  luminous  flame.  The  residue  is  then  taken  up 
with  a  small  amount  of  water  and  titrated  with  a  decinormal  solution 
of  hydrochloric  acid.  The  number  of  cubic  centimeters  employed  to 
bring  about  the  end-reaction,  multiplied  by  0.00365,  will  indicate 
in  terms  of  hydrochloric  acid  the  amount  of  organic  acids  which  were 
originally  present.  By  deducting  this  value  from  the  total  acidity, 
as  first  ascertained,  the  amount  of  physiologically  active  hydro- 
chloric acid  is  found. 

The  Ferments  of  the  Gastric  Juice  and  their  Proenzymes. 

In  the  gastric  juice  of  almost  all  vertebrate  animals  two  fer- 
ments are  constantly  found.  These  are  termed  pepsin  and  chy- 
mosin,  or  rennin,  and  are  supposedly  furnished  by  the  so-called 
adelomorphous  or  central  cells  of  the  gastric  glands.  This  has  been 
established  by  resecting  the  pyloric  end  of  the  stomach  and  convert- 
ing it  into  a  blind  pouch,  with  a  fistulous  opening  on  the  anterior 
abdominal  walls,  while  the  fundus  was  united  to  the  duodenum.  It 
was  then  noted  that  this  resected  portion  of  the  stomach,  in  which 
no  delomorphous  cells  are  found,  furnished  an  alkaline  and  markedly 


THE  GASTRIC  JUICE.  125 

viscid  secretion,  which  contained  pepsin  and  large  amounts  of  mucin, 
but  no  hydrochloric  acid.  A  reversal  of  the  experiment,  on  the 
other  hand,  in  which  the  fundus  was  thus  isolated,  showed  that 
here  both  pepsin  and  hydrochloric  acid  are  secreted.  As  this 
portion  of  the  stomach  contains  both  delomorphous  and  adelo- 
morphous cells,  the  conclusion  naturally  suggests  itself  that  the 
hydrochloric  acid  is  furnished  only  by  the  delomorphous  cells, 
while  the  pepsin— and  the  same  apparently  holds  good  for  chy- 
niosin — is  secreted  by  the  adelomorphous  cells.  The  latter  are 
hence  also  spoken  of  as  pepsin  calls,  while  the  delomorphous  cells 
are  similarly  termed  the  oxyntic  cells  of  the  stomach. 

As  in  the  case  of  the  ptyalin  of  the  saliva,  however,  it  appears 
that  the  ferments  in  question  do  not  exist  in  the  cells  as  such,  but  in 
the  form  of  proenzymes  or  zymogens,  which  are  termed  propepsin 
or  pepsinogen  and  prorennin  or  chymosinogen,  respectively. 

It  has  thus  been  shown  that  an  aqueous  extract  of  the  gastric 
mucosa  when  treated  with  1  per  cent,  of  soda,  and-  kept  at  a  tem- 
perature of  40°  C,  even  for  several  hours,  does  not  lose  its  digestive 
power,  and  can  be  rendered  physiologically  active  by  subsequently 
acidifving  with  hydrochloric  acid  to  from  0.2  to  0.3  per  cent,,  pro- 
viding that  the  animal  has  previously  fasted.  If  then,  however, 
such  artificial  gastric  juice  is  neutralized,  and  then  alkalinized  with 
soda  to  the  extent  of  only  0.5  per  cent.,  the  solution  is  rendered 
entirely  inactive  after  a  few  seconds,  when  warmed  to  the  tempera- 
ture of  the  body,  and  it  is  to  be  noted  that  the  subsequent  addition 
of  hydrochloric  acid  is  now  no  longer  capable  of  restoring '  the 
activity  of  the  enzyme.  This  demonstrates,  of  course,  that  while 
the  proenzyme  is  more  or  less  resistant  to  soda,  the  ferment  is  thereby 
rapidly  destroyed.  On  the  other  hand,  it  appears  that  pepsin  is 
more  resistant  to  the  influence  of  carbonic  acid  than  propepsin. 
Between  chymosin  and  its  zymogen  similar  relations  exist. 

Of  the  chemical  nature  of  the  proenzymes  and  the  manner  in 
which  they  arc  produced  by  the  cells,  practically  nothing  is  known. 
Nerve-influences,  no  doubt,  are  here  at  work,  as  in  the  case  of  the 
salivary  glands.  At  the  same  time  the  blood-supply  is  of  moment, 
and  we'  find  that  during  the  process  of  digestion  the  blood-vessels  are 
dilated,  and  that  the  venous  circulation  is  more  rapid  and  the  blood 
of  a  light-red  color.  But  as  in  the  salivary  glands,  it  is  certain  that 
the  height  of  the  blood-pressure  has  only  indirectly  to  do  with  the 
activitv  of  the  glands.  The  proenzymes  here,  as  there,  are  formed 
through  a  specific  activity  on  the  part  of  the  cells,  from  food- 
material  which   is  supplied  by  the  lymph. 

Whether  «.r  not  the  transformation  of  the  proenzymes  into  the 
corresponding  ferments  occurs  in  the  bodiesof  the  cells  has  not  been 
definitely  decided.  It  appears,  however,  that  in  the  majority  of 
animal-' which  have  been  examined  in  this  direction  the  glands 
secrete  only  the  proenzymes,  and  that  these  are  then  rendered 
physiologically  active  by  the  hydrochloric  acid  of  the  gastric  juice. 


126  THE  DIGESTIVE  FLUIDS. 

A  solution  of  propepsin,  which  may  be  obtained  by  macerating  in 
glycerin  the  mucous  membrane  of  a  fasting  animal,  is  thus  in  itself 
inert,  but  is  rendered  active  at  once  when  hydrochloric  acid  is  added 
to  the  extent  of  from  0.1  to  0.2  per  cent.  It  is  indeed  supposed 
that  pepsin,  which  in  itself  is  inactive  like  its  zymogen,  combines 
with  hydrochloric  acid,  which  alone  is  similarly  inert,  as  regards  its 
digestive  ability,  to  form  a  compound  acid,  the  so-called  pepsin- 
hydrochlorie  acid.  On  coming  in  contact  with  albuminous  mate- 
rial this  is  supposedly  decomposed,  with  the  formation  of  nascent 
hydrochloric  acid,  which  then  acts  as  the  active  digestive  principle, 
while  the  liberated  pepsin  combines  with  a  new  portion  of  hydro- 
chloric acid,  and  thus  serves  as  an  acid-carrier.  On  this  question, 
however,  a  uniformity  of  opinion  does  not  exist ;  still,  the  hypoth- 
esis is  an  attractive  one,  and  has  a  good  deal  in  its  favor.  If  we 
thus  regard  the  action  of  a  ferment  as  essentially  influencing  the 
rapidity  of  reaction,  the  action  of  the  weak  hydrochloric  acid  of 
the  gastric  juice  could  be  compared  to  the  effect  of  stronger  solutions 
upon  albumins  under  the  application  of  heat. 

Pepsin. — In  pure  form  pepsin  has  thus  far  not  been  obtained.  In 
the  form  we  are  able  to  isolate  the  substance,  it  occurs  as  an  amor- 
phous white  or  yellowish-white  powder  which  is  not  hygroscopic. 
It  is  soluble  in  water,  dilute  acids,  and  glycerin.  When  acidified 
with  hydrochloric  acid  to  the  extent  of  from  0.1  to  0.3  per  cent.,  it 
is  capable  of  dissolving  albumins,  with  the  formation  of  albumoses 
and  so-called  amphopeptone  (see  below).  This  can  readily  be 
demonstrated  as  follows :  an  artificial  gastric  juice  is  prepared  by 
dissolving  a  pinch  of  one  of  the  commercial  preparations  of 
pepsin  in  dilute  hydrochloric  acid  (0.1-0.2  per  cent.),  to  which  a 
flake  of  boiled  beef-fibrin  is  then  added.  The  mixture  is  kept  at  a 
temperature  of  about  40°  C,  when  it  will  be  noted  that  after  a  short 
time  the  fibrin  begins  to  swell  and  is  subsequently  dissolved.  In  the 
solution  which  thus  results  albumoses  and  peptones  can  be  demon- 
strated (see  page  183).  Other  acids,  such  as  sulphuric  acid,  nitric 
acid,  phosphoric  acid,  lactic  acid,  and  even  acetic  acid,  are  also  cap- 
able of  rendering  pepsin  physiologically  active,  but  much  larger 
amounts  of  these  are  necessary  to  bring  about  the  same  result.  In 
the  case  of  phosphoric  acid,  for  example,  an  acidity  of  10-12  pro 
mille  is  necessary.  Carbonic  acid  and  hydrocyanic  acid,  on  the 
other  hand,  are  without  effect.  Unlike  chymosin,  pepsin  does  not 
bring  about  coagulation  of  casein. 

In  neutral  and  alkaline  solutions  pepsin  is  inactive,  and,  as  has 
been  seen,  it  is  rapidly  destroyed  by  sodium  carbonate,  even  in  very 
small  amount.  Its  resistance  to  higher  temperatures  is  to  a  great 
extent  dependent  upon  the  reaction  of  its  solutions.  In  neutral 
solution  it  is  destroyed  at  55°  C.  ;  in  the  presence  of  0.2  per  cent, 
of  hydrochloric  acid  this  result  is  reached  only  at  65°  C,  and  in  the 
presence  of  peptones  and  certain  salts  a  temperature  of  70°  C  is 
necessary  to  bring  about  the  same  end.     In  the  dry  state,  on  the 


THE  GASTRIC  JUICE.  127 

other  hand,  the  ferment  may  be  heated  to  100°  C,  and  even  higher, 
without  being  destroyed.  At  temperatures  lower  than  40°  C.  pepsin 
is  still  active,  but  less  energetically  so,  and  at  0°  C.  its  action  ceases 
altogether. 

Alcohol  precipitates  pepsin  from  its  solutions  without  affecting  its 
subsequent  activity,  unless  the  exposure  has  been  prolonged.  Some 
of  the  salts  of  the  heavy  metals,  such  as  the  acetates  of  lead  and 
platinum  chloride,  as  also  tannic  acid,  magnesium  carbonate,  and 
ammonium  sulphate,  likewise  cause  the  precipitation  of  impure 
forms,  at  least,  but  are  without  effect  upon  the  ferment  itself.  Like 
the  albumins,  pepsin  docs  not  diffuse  through  animal  membranes. 

To  a  certain  extent  the  rapidity  of  digestion  is  dependent  upon 
the  amount  of  pepsin  that  is  available ;  but,  as  in  the  case  of  all 
ferments,  very  small  quantities  are  sufficient  to  effect  an  amount  of 
chemical  change  that  is  apparently  out  of  all  proportion  to  the 
amount  present.  Thus,  Petit  claims  that  a  pepsin  preparation, 
which  he  prepared  himself,  was  capable  of  dissolving  500,000  times 
its  weight  of  fibrin  in  seven  hours.  The  much  more  impure 
commericial  forms  are,  of  course,  far  less  active,  but  many  of  them 
possess  remarkable  digestive  power. 

The  ability  on  the  part  of  pepsin  to  digest  albumins  is,  however, 
limited ;  and  with  an  increase  in  the  amount  of  digestive  products 
formed,  its  activity  gradually  diminishes  and  finally  ceases.  This 
can  be  obviated  in  a  measure  by  removing  these  products  as  they 
are  formed,  and  may  be  artificially  accomplished  by  allowing  the 
digestion  to  take  place  in  a  parchment  tube  which  has  been  sus- 
pended in  dilute  hydrochloric  acid.  The  peptones  which  are  formed 
then  pass  from  the  tube  by  dialysis,  and  in  this  manner  digestion 
can  be  carried  much  further  than  under  other  conditions.  Com- 
plete digestion,  however,  may  even  then  not  be  achieved,  which 
is  probably  to  be  explained  by  the  reversible  action  of  the  fer- 
ment, as  already  described  (page  111). 

Little  is  known  of  the  chemical  nature  of  pepsin.  At  first 
sight,  it  is  apparently  related  to  the  albumins  ;  but  it  is  to  be  noted 
that  the  reactions  of  the  purer  forms  become  further  and  further  re- 
moved from  those  of  the  albumins  as  the  degree  of  purity  increases. 
An  analysis  of  a  fairly  pure  specimen  has  given  the  following 
result:  C,  47.75;  H,  6.5;  N,  14.24;  (),  30.20j  8,  1.31  per  cent.  < 
Specific  teste  for  the  demonstration  of  the  pepsin  of  the  gastric 
juice,  as  compared  with  other  proteolytic  ferments  which  similarly 
act  in  acid  solution-,  are  unknown.  As  a  result,  all  such  ferments 
have  been  designated  as  pepsin,  although  it  is  very  likely  that  they 
are  not  identical.  Such  ferments  have  been  observed  in  the  secre- 
tion of  the  glands  of  Brunner,  in  the  muscles,  the  kidneys,  the 
brain,  tin;   saliva,  ami    the   urine. 

Isolation  of  Pepsin. —  If  it  i-  merely  desired  to  obtain  an  effective 
solution  of  pep-in  without  regard  to  the  purity  of  the  substance, 
the    following   procedure    may    be  employed  : 


128  THE  DIGESTIVE  FLUIDS. 

v.  Wittich's  Method. — The  mucous  membrane  of  a  pig's 
stomach  is  carefully  dissected  off,  freed  from  mucus  by  washing 
with  water,  hashed,  rubbed  together  with  pure  quartz  sand,  and 
finally  treated  with  glycerin,  containing  0.1  per  cent,  of  hydrochloric 
acid,  in  the  proportion  of  10-20  grammes  for  1  part  of  mucous 
membrane.  The  mixture  is  kept  at  a  temperature  of  40°  C  for 
from  one  to  two  weeks,  when  it  is  filtered.  The  extract  which  is 
thus  obtained  may  then  be  used  for  experimental  purposes  by 
diluting  with  0.1-0.4  per  cent,  of  hydrochloric  acid,  in  the  propor- 
tion of  2-3  :  100. 

To  obtain  as  pure  a  preparation  as  possible,  Briicke's  method, 
or  one  of  its  many  modifications,  is  usually  employed.  But  it  is 
to  be  noted,  as  Gautier  has  pointed  out,  that  while  the  substance 
is  thus  obtained  in  large  amount,  it  is  but  little  active.  We  find, 
as  a  matter  of  fact,  that  a  small  flake  of  fibrin,  which  has  been 
previously  caused  to  swell  by  placing  it  in  dilute  hydrochloric  acid, 
dissolves  in  a  solution  of  Briicke's  pepsin  only  after  five  minutes, 
while  other  and  more  impure  preparations  are  decidedly  more 
effective.     Briicke's    method  is  as  follows  : 

Brucke's  Method. — The  mucous  membrane,  which  has  been 
carefully  dissected  off  and  washed  with  water,  is  placed  in  a  solu- 
tion of  dilute  phosphoric  acid  (12  pro  mille)  and  allowed  to  stand 
for  about  one  week.  By  this  time  digestion  has  usually  proceeded 
to  a  point  where  a  precipitate  is  no  longer  obtained  on  render- 
ing the  mixture  nearly  neutral  with  sodium  hydrate.  It  is  then 
neutralized  with  lime-water,  which  causes  a  precipitation  of  cal- 
cium phosphate,  while  the  pepsin  is  at  the  same  time  carried 
down  mechanically,  and  adheres  so  firmly  to  the  phosphates  that 
these  can  subsequently  be  washed  with  water  without  losing  any 
of  the  pepsin.  The  precipitate  is  then  dissolved  in  dilute  hydro- 
chloric acid,  and  the  solution  dialyzed  until  the  phosphates  and 
the  hydrochloric  acid  have  diffused  out.  The  remaining  solution, 
which  contains  the  pepsin,  is  now  treated  with  large  quantities  of 
strong  alcohol.  In  this  manner  the  ferment  is  precipitated,  and 
is  readily  collected  on  a  filter. 

Other  ferments,  such  as  ptyalin,  do  not  adhere  to  the  phosphates 
so  firmly  as  pepsin,  and  can  hence  be  removed  by  suspending  the 
precipitate  in  water  and  passing  a  current  of  air  through,  the  solu- 
tion for  some  time. 

Instead  of  dialyzing  the  acid  solution,  as  just  described,  and 
then  precipitating  with  alcohol,  it  is  also  possible  to  obtain  the 
ferment  by  treating  such  acid  solutions  with  a  concentrated  alco- 
holic-ethereal solution  of  cholesterin.  When  this  is  added  the 
cholesterin  is  immediately  thrown  down,  and  carries  the  pepsin 
with  it.  If  this  precipitate  is  then  collected  on  a  filter  and 
washed  with  alcohol,  the  cholesterin  is  dissolved,  while  the  ferment 
remains. 

Such  preparations,   however,  are    impure,  and  probably  contain 


THE  GASTRIC  JUICE.  129 

mucin-peptones,  which  have  resulted  during  the  digestion  of  the 
mucous  membrane. 

Purer  forms  may  possibly  be  obtained  according  to  Kuhxe's 
method.  To  this  end,  pigs'  stomachs  are  placed  in  large  quantities 
of  dilute  hydrochloric  acid,  and  are  allowed  to  digest  for  several 
week-.  As  soon  as  albumoses  are  only  present  in  comparatively  small 
amounts,  owing  to  their  transformation  into  peptones,  the  solution  is 
saturated  with  ammonium  sulphate.  In  this  manner  the  remaining 
albumoses,  and  with  them  the  pepsin,  are  precipitated.  This  mass 
is  then  further  treated  with  hydrochloric  acid  and  allowed  to 
stand,  when  a  further  portion  of  the  albumoses  is  transformed  into 
peptones.  By  repeating  this  process  all  the  albumoses  are  finally 
peptonized,  and  ultimately  a  nearly  pure  pepsin  is  thrown  down 
by  saturating  with  the  salt.  This  is  dissolved  in  water,  dialyzed, 
and  finally  precipitated  with  strong  alcohol,  and  rapidly  collected 
on  a  filter. 

Instead  of  using  the  stomachs  of  animals,  some  of  the  more 
active  commercial  products  may  be  directly  employed  and  purified, 
as  just  described. 

Such  preparations  give  scarcely  any  of  the  characteristic  color- 
reactions  of  the  albumins,  and  are  not  precipitated  by  tannic  acid. 

Quantitative  Estimation  of  Pepsin. — Accurate  methods  for  the 
quantitative  estimation  of  pepsin  are  not  available.  Relative  values, 
however,  can  be  obtained  by  the  following  method,  as  suggested  by 
Hammerschlag :  three  Esbach  tubes  (albuminimeters)  are  em- 
ployed. Tube  1  is  filled  to  the  mark  U  with  a  mixture  of  10  c.c. 
of  a  1  per  cent,  solution  of  serum-albumin  in  0.4  per  cent,  of 
hydrochloric  acid  and  5  c.c.  of  filtered  gastric  juice.  Tube  2, 
which  is  the  standard,  is  filled  to  the  same  mark  with  the  serum- 
albumin  solution,  but  receives  in  addition  0.5  gramme  of  pepsin. 
Tube  :>>  contain-  merely  10  c.c.  of  the  albumin  solution  and  5  c.c. 
of  di -til led  water.  Esbach' 8  reagent,  which  consists  of  10  grammes 
of  picric  acid  and  20  grammes  of  citric  acid,  dissolved  in  1000 
c.c.  of  distilled  water,  is  then  added  to  each  tube  to  the  mark  K. 
A  tier  twenty-four  hours  the  amount  of  precipitate  is  read  off 
and  the  difference  between  tubes  1  and  3  compared  witli  that  of 
tube  2. 

Pepsinogen. — The  presence  <»f  pepsinogen  in  the  gastric  juice 
'•an  be  ascertained  only  when  hydrochloric  acid  is  absent,  as  it  is 
otherwise  transformed  into  the  active  enzyme.  Its  occurrence,  as 
such,  i-  hence  essentially  a  pathologic  phenomenon, and  indicates  the 
absence  of  free  hydrochloric  acid.  Hut  while  the  latter  may  be 
absent  in  many  diseases  which  are  not  associated  with  structural 
abnormalities  of  the  gastric  mucous  membrane,  pepsinogen, and  con- 
sequently also  pepsin,  are  found  lacking  only  in  disease  of  the 
stomach  it-elf  mid  when  complete  atrophy  of  the  glandular  struct- 
ures  ha-  occurred. 

Test  for  Pepsinogen. — Specimens  of  gastric  juice  in  which  pepsin- 


130  THE  DIGESTIVE  FLUIDS. 

ogen  only  is  present  are  incapable  of  digesting  albumins.  In  such 
cases,  as  I  have  just  said,  hydrochloric  acid  is  absent.  If  then 
the  solution  is  acidified  to  the  extent  of  from  0.1  to  0.3  per 
cent.,  and  the  dissolution  of  a  flake  of  boiled  beef-fibrin  now  occurs, 
the  presence  of  the  zymogen  may  be  inferred. 

Quantitative  Estimation  of  Pepsinogen. — The  determination  of  the 
absolute  quantity  of  pepsinogen  in  the  gastric  juice,  as  that  of  pep- 
sin, is  not  possible.  Relative  values,  however,  may  be  obtained  by 
the  following  method,  as  suggested  by  Boas  :  To  this  end,  specimens 
of  the  gastric  juice  are  variously  diluted  with  distilled  water  in  the 
proportion  of  1  :  5,  1  :  10,  1  :  20,  etc.  A  known  quantity  of  coagu- 
lated egg-albumin  or  serum-albumin  is  then  added  to  each  tube, 
as  also  1  or  2  drops  of  a  0.3  per  cent,  solution  of  hydrochloric  acid, 
for  every  10  c.c.  The  tubes  are  kept  at  a  temperature  of  about 
40°  C,  when  the  degree  of  dilution  is  noted  at  which  the  albumin  is 
still  dissolved.  The  greater  the  degree  of  dilution  at  which  this 
occurs,  of  course  the  greater  is  the  amount  of  pepsinogen — that  is, 
of  pepsin — present. 

Should  it  be  desired  to  exclude  definitely  the  presence  of  pepsin 
and  pepsinogen  in  the  stomach,  200  c.c.  of  a  decinormal  solution  of 
hydrochloric  acid  are  introduced  into  the  organ  through  a  tube,  and 
the  remaining  liquid  removed  after  one-half  hour.  If  this  fluid 
then  contains  no  pepsin,  the  absence  of  the  zymogen  may  also  be 
inferred. 

Chymosin. — Chymosin,  or  rennin,  like  pepsin,  is  secreted  by  the 
central  cells  of  the  gastric  glands  in  the  form  of  a  pro-enzyme,  which, 
like  the  pepsinogen,  is  then  transformed  into  the  corresponding  fer- 
ment by  the  hydrochloric  acid  of  the  gastric  juice.  It  is  to  be 
noted,  however,  that  calcium  chloride  or  any  other  soluble  calcium 
salt  is  likewise  capable  of  bringing  about  this  result.  The  specific 
action  of  chymosin  is  exerted  upon  milk  or  lime-containing  solutions 
of  casein,  which  are  coagulated  in  neutral  and  even  feebly  alkaline 
solutions.  Unlike  pepsin,  chymosin  is  not  a  proteolytic  ferment, 
and  its  action  ceases  with  the  formation  of  paracasein.  It  is  there- 
fore surprising  to  note  that  chymosin  is  found  not  only  in  the 
stomachs  of  mammals,  but  also  in  other  vertebrate  animals,  and 
even  in  certain  plants,  where  casein  as  a  food-stuff  certainly  does  not 
enter  into  consideration.  Our  knowledge  of  ferments  in  general,  how- 
ever, is  as  yet  very  defective,  and,  as  a  matter  of  fact,  we  are  acquainted 
only  with  the  more  manifest  reactions  of  these  bodies,  while  it  is 
quite  possible  that  they  possess  other  important  properties  of  which 
we  are  now  in  ignorance.  It  is  conceivable,  moreover,  that  differ- 
ent varieties  of  chymosin  exist,  which,  as  a  class,  are  all  capable 
of  coagulating  casein,  but  wrhich  differ  from  each  other  in  other 
respects  and  serve  other  purposes. 

While  chymosin  is  also  active  in  feebly  acid  solution,  it  is  gradu- 
ally destroyed  at  a  temperature  of  from  37°  to  40°  C.  when  exposed 
to  the  action  of  gastric  juice  containing  0.3  per  cent,  of  hydrochloric 


THE  GASTRIC  JUICE.  131 

acid.  The  ferment  is  here  apparently  digested  by  the  pepsin,  and  it 
is  thus  easily  possible  to  obtain  solutions  of  pepsin  which  are  alto- 
gether free  from  chymosin.  In  neutral  solution  it  is  more  resistant, 
and  can  be  heated  to  a  temperature  of  50°  C. ;  at  70°  C,  however, 
it  becomes  permanently  inactive.  In  its  dry  state,  on  the  other 
hand,  it  can  be  heated  to  110°  C.  without  losing  its  activity.  Alka- 
lies when  present  beyond  traces  destroy  the  substance,  as  they  do 
pepsin.  Like  all  other  ferments,  it  is  capable  of  effecting  an  exten- 
sive reaction,  even  when  present  in  small  amount.  The  quantity  of 
ferment  contained  in  1  gramme  of  the  dried  and  pulverized  mucous 
membrane  of  the  fourth  stomach  of  the  calf,  when  dissolved  in 
water,  is  thus  capable  of  coagulating  200  liters  of  milk  in  one 
minute  at  a  temperature  of  50°  C 

Of  the  chemical  nature  of  chymosin,  nothing  is  known ;  but,  as 
in  the  case  of  pepsin,  the  purer  preparations  do  not  give  the  usual 
reactions  of  albumins.  It  is  precipitated  from  its  neutral  solutions 
by  subacetate  of  lead,  while  the  acetate  and  tannic  acid  are  without 
effect.  Alcohol  likewise  precipitates  the  ferment  and  gradually 
renders  it  inactive.     Like  pepsin,  it  is  not  dialyzable. 

Under  normal  conditions  chymosin  is  always  present  in  the  gastric 
juice  of  man.  In  certain  diseases  of  the  stomach,  however,  which 
are  associated  with  the  death  of  its  glandular  elements,  the  ferment,  as 
also  its  zymogen,  is  lacking. 

Tests  for  Chymosin  and  Chymosinogen. — To  test  for  the  presence 
of  chymosin,  5  or  10  c.c.  of  milk  are  treated  with  a  few  drops  of 
the  filtered  gastric  juice  and  kept  at  a  temperature  of  from  37°  to 
40°  C.  If  coagulation  occurs  within  ten  or  fifteen  minutes,  the 
presence  of  chymosin  may  be  assumed.  Should  the  gastric  juice, 
however,  be  markedly  acid,  it  is  necessary  first  to  neutralize  it  with 
sodium  hydrate. 

To  test  for  chymosinogen,  the  milk  is  treated  with  2-3  c.c.  of  a 
1  per  cent,  solution  of  calcium  chloride,  and  10  c.c.  of  filtered 
gastric  juice  which  has  been  rendered  feebly  alkaline  with  sodium 
hydrate.  The  mixture  is  kept  at  a  temperature  of  from  37°  to  40° 
(\,  when  in  the  presence  of  the  zymogen  a  thick  cake  of  casein  is 
formed  within  a  few  minutes. 

Isolation  of  Chymosin. — To  isolate  chymosin  in  comparatively 
pure  form,  the  following  method,  as  suggested  by  Ilammarsten,  may 
be  employed  :  The  mucous  membrane  of  the  fourth  stomach  of  the 
calf  is  carefully  dissected  off,  washed  with  water, and  extracted  with 
an  o.l  percent,  solution  "I'  hydrochloric  acid,  as  already  described. 
The   infusion    is   then    neutralized    mid    repeatedly   shaken    with 

powdered  magnesium  carbonate  until  the  pepsin  has  been  removed. 
The  filtrate  is  treated  with  subacetate  of  lead,  the  precipitate 
decomposed  with  very  dilute  sulphuric  acid,  and  the  acid  filtrate 
further  precipitated  with  an  aqueous  solution  of  stearin  soap.  The 
fermenl  i-  thus  thrown  down  together  with  the  fatty  acids,  from 
which  it  i-  then  separated  by  suspending  the  precipitate  in  w;it<  r 


132  THE  DIGESTIVE  FLUIDS. 

and  extracting  the  fatty  acids  with  ether.  The  chymosin  remains  in 
aqueous  solution,  and  may  now  be  precipitated  with  strong  alcohol. 
It  is  then  rapidly  collected  on  a  filter  and  dried. 

Quantitative  Estimation  of  Chymosin  and  Chymosinogen. — As  in 
the  case  of  pepsin  and  pepsinogen,  relative  values  only  can  be 
obtained.  The  gastric  juice  is  neutralized  with  a  very  dilute  solu- 
tion of  sodium  hydrate.  Tubes  are  then  prepared,  containing  5  or 
10  c.c.  of  the  gastric  juice,  variously  diluted  in  the  proportion  of 
1  :  10,  1  :  20,  1  :  30,  etc.,  to  which  an  equal  volume  of  neutral  or 
amphoteric  milk  is  further  added.  These  tubes  are  kept  at  a 
temperature  of  from  37°  to  40°  C,  when  the  degree  of  dilution  is 
noted  at  which  coagulation  still  occurs.  Under  normal  conditions 
a  positive  reaction  can  thus  be  obtained  in  man  with  a  degree  of 
dilution  varying  between  1  :  30  and  1  :  40. 

In  the  case  of  the  zymogen,  the  gastric  juice  is  rendered  feebly 
alkaline,  when  tubes  are  prepared  as  just  described.  Normally  a 
positive  reaction  can  thus  still  be  obtained  with  a  dilution  varying 
between   1  :  100  and  1  :  150. 

Other  Constituents  of  the  Gastric  Juice. — Of  other  con- 
stituents, the  gastric  juice  normally  contains  traces  of  sulpho- 
cyanides,  which  are  secreted  by  the  stomach  itself;  a  variable 
amount  of  mucin  ;  a  small  amount  of  coagulable  albumin,  or,  if  the 
fluid  has  stood  for  some  time,  a  corresponding  quantity  of  albumoses 
or  peptones,  and,  as  already  shown,  certain  mineral  salts. 

The  gases  which  are  found  in  the  stomach  have  in  part  been 
swallowed  with  the  food.  A  small  portion  is  further  referable  to 
eructations  from  the  duodenum,  while  a  third  portion  is  probably 
secreted  by  the  stomach  itself.  This  is  true  more  especially  of 
the  carbon  dioxide,  and  Schierbeck  has  shown  that  the  tension 
of  this  gas  gradually  increases  from  30  to  40  Hgmm.  while 
fasting,  to  130  to  140  Hgmm.  during  the  process  of  digestion, 
and  is  apparently  directly  proportionate  to  the  acidity  of  the 
gastric    juice. 

An  idea  of  the  relative  amounts  of  the  gases  which  are  normally 
found  in  the  stomach  may  be  formed  from  the  accompanying  table, 
which  is  taken  from  Planer : 

Man.  Dog. 

Vegetable  diet.  Veg.  diet.        Meat  diet, 

vol.  per  cent.  vol.  per  cent.    vol.  per  cent. 

Carbon  dioxide 20.79-33.83  32.9  25.2 

Oxygen 0.37     .    .  0.8  6.1 

Nitrogen 72.50-33.22  66.3  68.7 

Hydrogen 6.71-27.58 

Other  gases,  such  as  marsh  gas,  olefiant  gas,  ammonia,  and  hy- 
drogen sulphide,  are  found  only  under  pathologic  conditions,  and 
are  referable  to  certain  fermentative  and  putrefactive  changes  which 
take  place  in  the  ingested  food. 


THE  PANCREATIC  JUICE.  133 


THE  PANCREATIC  JUICE. 


As  has  been  pointed  out,  the  digestive  glands  which  have  so  far 
been  considered  are  not  essential  to  the  maintenance  of  life.  The 
salivary  glands  and  the  stomach,  moreover,  can  in  certain  animals 
be  eliminated  without  seriously  interfering  with  the  process  of 
digestion,  and  the  ferments  which  in  man  are  secreted  by  these 
structures  are  in  many  animals  absent,  The  pancreas,  on  the  other 
hand,  either  as  such  or  as  a  so-called  hepatopancreas,  is  found  in  all 
vertebrate  and  invertebrate  animals  in  which  the  process  of  diges- 
tion is  carried  on  in  a  well-defined  digestive  tube.  In  many, 
indeed,  it  represents  the  only  digestive  gland  of  the  body.  Its 
removal,  even  in  the  higher  animals,  invariably  leads  to  death.  In 
dogs,  in  which  this  operation  has  been  repeatedly  performed,  and  in 
which  life  may  go  on  for  a  few  weeks  thereafter,  it  has  been 
observed  that  as  a  result  of  such  interference  the  resorption  of  fats 
is  seriously  impeded,  so  that  practically  all  that  has  been  ingested 
reappears  in  the  feces.  In  the  case  of  the  albumins,  it  is  similarly 
found  that  but  44  per  cent,  is  absorbed,  and  of  the  ingested  starches 
from  20  to  40  per  cent,  is  eliminated  as  such.  Analogous  results 
are  obtained  in  the  human  being  where  atrophy  of  the  pancreas  is 
at  times  observed.  As  a  consequence,  rapid  emaciation  occurs,  and,  as 
has  been  stated,  death  ultimately  results.  It  appears,  however,  that 
the  fatal  issue  in  these  cases  is  not  exclusively  referable  to  impaired 
nutrition  as  a  result  of  defective  absorption.  It  is,  indeed,  possible 
to  counteract  this  effect  by  administering  a  sufficient  amount  of  raw 
pancreas  together  with  the  food,  whereby  the  resorption  of  both  fats 
and  albumins  is  greatly  improved.  Death,  however,  takes  place  never- 
theless. It  is  thus  apparent  that  besides  its  digestive  function  the 
pancreas  must  play  an  additional  and  important  rdle  in  the  metab- 
olism of  the  animal  body.  We  find,  as  a  matter  of  fact,  that  fol- 
lowing the  extirpation  of  the  pancreas  in  dogs  a  severe  form  of  dia- 
betes  rapidly  develops,  and  is  accompanied  by  the  appearance  of 
acetone,  diacetic  acid,  and  at  times  of  ,9-oxvbutyric  acid  in  the  urine. 
That  this  is  not  due  to  suspension  of  the  pancreatic  digestion  can  be 
proved  in  various  ways.  If  the  animal  thus  receives  an  adequate 
amount  of  raw  pancreas  together  with  its  food,  the  absorption  of 
albumins  and  fats  is,  as  just  stated,  greatly  increased,  while  the 
diabetes  persists.  It  has  been  further  noted  thai  ligation  of  the 
secretory  dud  does  not  lead  to  the  appearance  of  sugar  in  the  urine, 
and  that  the  diabetes  continues  after  extirpation  even  when  no  food 
i-  consumed  for  several  days.  The  conclusion  hence  suggests  itself 
ihat  the  pancreas,  like  the  thyroid,  the  adrenal  body,  and  other 
glands,  probably  furnishes  an  internal  secretion  also,  which  in  some 
manner,  as  yet  unknown,  controls  the  metabolism  of  glucose  within 
the  animal  body.  Arthaud  and  Butte,  it  is  true,  claim  that  diabetes 
doe-  n,,i  follow   ligation  of  the  pancreatic  veinsj  bul  it  can  readily 


134  THE  DIGESTIVE  FLUIDS. 

be  imagined  that  in  such  cases,  and  perhaps  even  under  normal 
conditions  the  internal  secretion  of  the  gland  is  removed  through 
the  lymph-channels.  It  has  been  shown,  moreover,  that  diabetes 
does  not  occur  after  extirpation  of  the  pancreas  if  a  piece  of  the 
gland  has  been  previously  transplanted  under  the  skin. 

Of  the  nature  of  the  substance  or  substances  which  are  thus 
secreted  by  the  pancreas,  and  in  the  presence  of  which  the  carbo- 
hydrate metabolism  continues  in  a  normal  manner,  Ave  know  nothing. 
According  to  Lepine  and  his  school,  the  gland  is  supposed  to  furnish 
a  glucolytic  ferment,  which  brings  about  oxidation  of  the  sugar  in  the 
tissues,  and  it  will  be  seen  that  such  a  ferment  can  actually  be  demon- 
strated in  the  blood.  The  time  has  not  come,  however,  when  we 
can  speak  definitely  on  this  subject.      / 

The  secretion  of  the  pancreatic  digestive  fluid,  like  that  of  the 
saliva,  is  partly  under  the  control  of  cerebrospinal  nerve-fibres,  which 
are  derived  from  the  vagus,  and  partly  of  sympathetic  fibres.  The 
material  from  which  the  secretion  is  elaborated  through  the  specific 
activity  of  the  glandular  cells  is  obtained  from  the  lymph,  and  ulti- 
mately, of  course,  from  the  blood.  In  carnivorous  animals,  in  which 
the  secretion  of  the  pancreas  is  intermittent  and  dependent  upon  the 
ingestion  of  food,  we  accordingly  find  that  in  its  stage  of  activity  the 
gland  assumes  a  bright  rose-color,  and  is  much  increased  in  size, 
while  in  the  resting  stage  it  is  pale  and  shrunken. 

General  Properties. — Fresh  pancreatic  juice  can  be  obtained 
only  by  establishing  an  artificial,  fistula  in  the  pancreatic  duct.  But 
as  the  least  interference  with  the  integrity  of  the  gland  leads  at  once 
to  the  secretion  of  an  abnormal  fluid,  great  care  must  be  exercised 
to  operate  as  gently  and  rapidly  as  possible.  It  is  best  to  do  so 
about  three  hours  after  the  animal  has  received  a  large  meal.  For 
a  short  while  at  least,  a  normal  secretion  can  then  be  obtained.  This 
represents  a  clear,  thick,  colorless  and  odorless,  very  concentrated 
fluid,  of  a  strongly  alkaline  reaction,  which  actively  digests  albumins, 
inverts  starches  and  the  more  complex  sugars,  and  emulsifies  fats. 
After  a  variable  length  of  time,  however,  the  secretion  becomes 
thinner,  more  deficient  in  solids,  and  otherwise  altered,  so  that  it 
can  scarcely  be  regarded  as  normal. 

Specific  Gravity. — The  specific  gravity  of  the  pancreatic  juice 
varies  between  1.008  and  1.010,  corresponding  in  man  to  the  pres- 
ence of  from  2.5  to  7  per  cent,  of  solids.  When  kept  for  a  few 
hours  at  ordinary  temperatures  it  loses  its  viscosity  and  transparency, 
and  rapidly  undergoes  putrefaction.  Crystals  are  then  deposited 
which  consist  of  leucin  and  tyrosin  ;  they  result  from  the  digestion 
and  subsequent  decomposition  of  contained  albumins.  To  prevent 
these  changes,  the  secretion  must  be  placed  on  ice  at  once,  or  treated 
with  chloroform- water  or  a  similar  antiseptic  solution.  Owing  to 
the  presence  of  the  large  amounts  of  albumin  Avhich  the  pancreatic 
juice  contains,  the  liquid  coagulates  to  a  dense  mass  when  heated  to 
a  temperature  of  74°  C.     On  cooling  to  0°  C,  or  when  dropped  into 


THE  PANCREATIC  JUICE.  135 

water,  a  clot  is  formed,  which  redissolves  on  warming  the  solution  or 
on  adding  an  excess  of  sodium  chloride. 

Amount. — The  amount  of  pancreatic  juice  which  is  secreted  in 
the  twenty-four  hours  is  variable,  and  largely  dependent  upon  the 
quantity  and  quality  of  the  food  ingested.  From  permanent  fistula? 
45-100  grammes  are  supposedly  secreted  pro  kilogramme  of  the 
animal.  This  figure,  however,  is  no  doubt  too  high,  and  Bidder  and 
Schmidt  have  estimated  that  under  normal  conditions  the  secretion 
probably  does  not  exceed  5  grammes  pro  kilo. 

In  a  recent  case  of  pancreatic  fistula,  observed  in  man,  Pfaff  re- 
ports that  ()00  c.c.  of  the  secretion  were  obtained  in  twenty-four 
hours.  This  figure  thus  more  nearly  corresponds  to  the  results 
reached  by  Bidder  and  Schmidt. 

Chemical  Composition. — A  general  idea  of  the  chemical  composi- 
tion of  the  pancreatic  juice  of  the  dog  may  be  formed  from  the 
accompanying  analyses,  which  are  taken  from  C.  Schmidt  and 
Kriiger.  In  these  the  essential  points  of  difference  which  exist 
between  the  normal  fluid,  as  compared  with  the  secretion  obtained 
from  permanent  fistuhe,  are  well  shown.  I  have  further  added  two 
analyses  of  human  pancreatic  juice  which  were  made  by  Herter  and 
Zawadsky.  In  Herter's  case  an  accumulation  of  the  secretion  had 
taken  place  in  the  duct,  owing  to  a  pressure-stenosis,  which  was  pro- 
duced by  a  carcinomatous  growth  ;  while  Zawadsky's  material  was 
obtained  from  a  pancreatic  fistula  which  had  remained  after  removal 
of  a  pancreatic  tumor. 

Of  the  two  secretions,  the  first  is  manifestly  abnormal  (see  below), 
while  the  analytical  results  in  the  second  case  conform  closely  with 
the  figures  which  were  obtained  by  Schmidt  in  what  may  be  regarded 
as  the  normal  pancreatic  juice  of  the  dog. 

I  have  finally  appended  an  analysis  of  the  contents  of  a  pancreatic 
cyst,  which  in  its  general  composition  resembles  the  secretion  ob- 
tained from  permanent  fistuke  in  animals.  Trypsin,  however,  was 
absent. 

Secretion  from  a  tem-  Secretion  from  a  per- 

porary  fistula  of  the  manent  fistula  of  the 
dog.  dog. 

(C  Schmidt.)  (Kruger.) 

Water 900.8 980.11 

Solids 99.2 19. CO 

Albumins,  peptones,  and }  ro  .,  q  ,o 

ferments.  ( 

Organic    matter,    comprising  "J 

lenein,  tyrosin,   zantbins,  [     30.4 3.3 

goaps,  and  fats.  J 

Mineral  constituents 8.8     3.57 

Sodium  chloride 7.35 0.93 

Potassium  chloride    ....  0.02 0.07 

Phosphate  of  calcium  .   .    .  0.41 0.01 

Phosphate  of  magnesium  .    .  0.12 0.02 

Lime  and  magnesia   ....  0.32 2.63 


136  THE  DIGESTIVE  FLUIDS. 


Human.  Human. 

(Herter's  case.)  (Zawadsky's  case.) 

Water 975.9 864.05 

Solids 24.2 135.95 

Peptones  and  enzymes  )  .. ..  _ 

(no  albumin.)  /   •    •    •    ■     li-° 92.05 

Alcoholic  extract 6.4 43.90 

Mineral  ash 6.2 3.44 

Analysis  of  the  Contents  of  a  Pancreatic  Cyst  (Lenarcic), 

Water 928.1 

Solids 17.9 

Organic  matter 10.05 

Mineral  ash 7.85 

The  Ferments  and  their  Zymogens. 

The  most  important  constituents  of  the  pancreatic  juice  are  the 
ferments,  of  which  five  different  forms  are  said  to  be  present. 
These  are  trypsin,  steapsin,  an  amylolytic  ferment  (which  is  said 
to  be  identical  with  the  salivary  ptyalin),  maltase,  and  chymosin. 
Like  the  ferments  which  are  furnished  by  the  salivary  glands  and 
the  central  cells  of  the  gastric  glands,  these  enzymes  occur  also  in  the 
pancreatic  cells  in  the  form  of  zymogens,  which  are  subsequently 
transformed  into  the  active  ferments.  If  a  fresh  pancreas  is  thus 
extracted  with  glycerin,  it  will  be  noted  that  the  resulting  extract 
has  no  proteolytic  properties  whatever,  while  an  extract  obtained 
after  the  gland  has  been  hashed  and  exposed  to  the  air  for  some 
time,  or  an  aqueous  extract  of  the  fresh  gland,  digests  albumins 
with  ease.  If  the  fresh  gland,  further,  is  hashed  and  briefly  treated 
with  a  1  per  cent,  solution  of  acetic  acid,  and  then  extracted  with 
glycerin,  an  active  preparation  is  obtained  at  once.  Similar  results 
are  reached  in  the  case  of  the  pancreatic  ptyalin.  If  a  fresh  pancreas 
is  thus  repeatedly  extracted  with  glycerin  until  ptyalin  no  longer 
passes  into  solution,  and  the  gland  is  then  washed  in  water  and  left 
exposed  to  the  air  for  a  short  time,  an  additional  amount  of  the 
ferment  can  be  obtained  by  further  extraction  with  the  same  reagent. 
In  what  manner  the  transformation  of  trypsinogen,  ptyalinogen, 
steapsinogen,  and  the  other  zymogens,  into  the  corresponding  fer- 
ments is  normally  effected,  is  unknown.  But,  as  has  been  seen,  this 
can  be  brought  about  artificially  through  the  influence  of  water, 
dilute  acetic  acid,  and  probably  also  through  the  activity  of  acid- 
forming  bacteria  or  the  oxygen  of  the  air. 

Of  the  chemical  composition  of  the  zymogens  we  know  little,  but 
it  appears  that  they  are  of  an  albuminous  nature  and  of  a  more 
complex  composition  than  the  ferments  themselves.  On  decomposi- 
tion trypsinogen  thus  yields  the  corresponding  ferment  and  an 
unknown  albuminous  substance.  Of  the  origin  of  the  zymogens, 
and  of  the  manner  in  which  they  are  produced  in  the  cells,  we  know 
nothing. 

Trypsin. — Trypsin  is  the  most    important  proteolytic  ferment 


THE  PANCREATIC  JUICE.  137 

which  is  found  in  the  animal  world,  and  in  its  action  on  albumins  is 
fully  capable  of  replacing  pepsin  when  this  is  absent.  Its  digestive 
power,  moreover,  is  much  more  extensive  than  that  of  pepsin,  and  it 
is  further  capable  of  decomposing  albumins  to  amido-acids  and 
hexon  bases.  Its  hydrolytic  effect  may  thus  be  compared  to  the 
action  of  strong  mineral  acids  under  the  application  of  heat.  ^This 
explains  also  the  fact  that  leucin  and  tyrosin  are  so  frequently  found 
in  the  pancreatic  juice.  The  extensive  digestive  activity  of  trypsin 
is  well  shown  when  the  gland  is  finely  hashed  and  treated  with  a 
large  amount  of  chloroform- water,  so  as  to  guard  against  putrefac- 
tive changes.  When  kept  at  a  temperature  of  40°  C.  autodigestion 
rapidly  takes  place,  and  after  several  days  it  will  be  noted  that  while 
trypsin  is  -till  present  in  its  full  activity,  the  other  ferments  have 
disappeared.  Together  with  the  various  albumins  of  the  gland  they 
have  apparently  been  digested  by  the  more  powerful  ferment. 

While  trypsin  acts  most  energetically  in  feebly  alkaline  or  neutral 
solutions  (0.25-1.0  per  cent,  of  sodium  carbonate),  it  is  also  capable 
of  digesting  albumins  in  slightly  acid  media,  providing  that  the 
acidity  is  not  due  to  the  presence  of  a  free  mineral  acid;  the  diges- 
tive process  is  under  such  conditions,  however,  much  less  active. 
Free  mineral  acids  rapidly  destroy  the  ferment.  Its  optimum  tem- 
perature lies  between  37b  and  40°  C.  In  neutral  solution  it  is 
destroyed  at  45°  C,  while  in  feebly  alkaline  media,  and  especially 
in  the"  presence  of  albumoses  and  certain  ammoniacal  salts,  it  can  be 
heated  somewhat  higher  without  impairment  of  its  digestive  power. 
A-  in  the  case  of  pepsin,  the  digestive  effect  of  trypsin  is  to  a 
certain  extent  dependent  upon  the  amount  of  the  ferment  present, 
and  here  as  there  the  action  of  the  enzyme  ultimately  ceases 
when  the  digestive  products  accumulate  beyond  a  certain  amount. 
Impure  extracts  can  be  prepared,  as  lias  been  pointed  out,  by 
extracting  the  gland  with  glycerin  after  it  has  been  left  exposed  to 
the  air.  The  purer  forms,  on  the  other  hand,  which  can  be  obtained 
according  to  Iviilme's  method  (see  below),  are  insoluble  in  glycerin 
and  alcohol,  but  soluble  in  water. 

Of  the  chemical  nature  of  trypsin  little  is  known.  In  acid  solu- 
tion it  is  coagulated  by  heat,  and,  according  to  Kiihne,  decomposed 
into  an  albumin  and  peptone.  An  analysis  of  a  fairly  pure  prepara- 
tion has  given  the  following  results:  carbon,  02.75  percent.;  hydro- 
gen, 7.51;  nitrogen,  16.55 ;  oxygen  pins  sulphur,  2:5.19  (Loew). 
Its  composition  is  thus  very  similar  to  that  of  the  peptones,  and  it  is 
hence  possible  that,  unlike  the  other  digestive  ferments  which  we 
have  thus  far  considered,  trypsin  may  be  an  albuminous  substance. 
Test  for  Trypsin. — The  tesl  for  trypsin  resolves  itself  into  the 
demonstration  of  the  presence  of  a  proteolytic  ferment  which  is 
capable  of  digesting  albumins  in  alkaline  solution,  with  the  ultimate 
formation  of  amido-acids.  To  this  end,  the  solution  in  question  is 
rendered  alkaline  with  sodium  carbonate  to  the  extent  of  from  0.25 
,,,   i.i,   per  cent.     A   -mall   flake  of  fibrin  is  then  added  and  an 


138  THE  DIGESTIVE  FLUIDS. 

amount  of  thymol  sufficient  to  saturate  the  solution,  so  as  to  guard 
against  putrefactive  processes.  The  mixture  is  kept  at  a  tempera- 
ture of  40°  C.  If  the  fibrin  is  then  dissolved  and  leucin  or  tyrosin 
can  be  demonstrated  in  the  resulting  solution,  the  presence  of  trypsin 
may  be  inferred.  To  this  end,  it  is  only  necessary  to  evaporate  the 
solution  to  a  thick  syrup  and  to  examine  this  microscopically  (see 
page  188).  Should  the  solution  to  be  tested  contain  a  free  mineral 
acid,  or  larger  amounts  of  organic  acids,  no  result  will,  of  course,  be 
obtained,  as  the  trypsin  has  then  been  destroyed. 

Isolation  of  Trypsin. — Unless  it  is  desired  to  obtain  trypsin  in  as 
pure  a  form,  as  possible,  alkaline  solutions  of  the  common  pancreatin 
preparations  which  are  sold  in  the  shops  can  be  used  for  experimental 
purposes.  Otherwise  the  method  of  Kiihne,  as  modified  by  Gautier, 
should  be  employed  :  The  fresh  pancreas  is  finely  hashed  and 
washed  with  ice-cold  water,  to  which  1  pro  mille  of  salicylic  acid 
has  been  added.  After  four  hours  the  mass  is  treated  with  a 
large  amount  of  a  5  pro  mille  solution  of  sodium  carbonate, 
containing  an  excess  of  thymol,  and  is  kept  for  twelve  hours  at  a 
temperature  of  from  37°  to  40°  C.  The  acid  and  alkaline  extracts 
are  then  mixed,  treated  with  0.5  per  cent,  of  sodium  carbonate, 
filtered,  feebly  acidulated  with  acetic  acid,  and  saturated  with 
ammonium  sulphate  in  substance.  The  precipitate  which  then 
separates  out  contains  all  the  trypsin.  It  is  dissolved  in  water, 
filtered,  and  dialyzed,  so  as  to  remove  the  salt  which  is  still  present, 
as  also  traces  of  peptones  and  leucin.  The  resulting  solution  is 
concentrated  at  a  very  low  temperature,  and  the  trypsin  finally  pre- 
cipitated with  strong  alcohol.  It  is  then  rapidly  filtered  off  and 
dried.  The  amount  of  material  which  can  thus  be  obtained  from 
one  gland  is  always  very  small,  but  sufficient  to  digest  a  large 
quantity  of  albumin  when  dissolved  in  about  100  c.c.  of  water. 

The  so-called  dry  pancreas  of  Kiihne,  from  which  trypsin  can 
likewise  be  obtained,  and  which  is  also  used  in  digestive  experiments 
as  such,  is  prepared  by  extracting  the  fresh  gland  with  alcohol  and 
subsequently  with  ether  until  it  is  free  from  fats.  The  remaining 
material,  which  contains  the  active  ferment,  is  then  dried  and  pul- 
verized, and  can  be  kept  in  this  form  indefinitely. 

The  Amylolytic  Ferment  of  the  Pancreatic  Juice  (Amylop- 
sin). — The  amylolytic  ferment  of  the  pancreatic  juice  is  by  many 
thought  to  be  identical  with  the  ptyalin  of  the  saliva.  It  can  be 
isolated,  according  to  Gautier's  method,  from  an  aqueous  infusion  of 
the  fresh  gland  that  has  remained  exposed  to  the  air  for  about 
twenty-four  hours,  as  already  described.  To  demonstrate  its  action, 
a  few  cubic  centimeters  of  such  an  infusion,  or  a  glycerin  extract  of 
the  gland,  are  added  to  a  small  amount  of  starch  paste  and  kept  at  a 
temperature  of  40°  C.  The  mixture  is  then  tested  for  the  presence 
of  maltose,  as  already  described. 

Steapsin. — It  has  long  been  known  that  the  pancreatic  juice  pos- 
sesses the  power  of  emulsifying  fats,  and  of  decomposing  these  into 


THE  PANCREATIC  JUICE.  139 

glycerin  and  the  corresponding  fatty  acids.  While  this  phenomenon 
has  by  some  been  referred  to  the  action  of  bacteria,  others  hold  that 
it  is  dependent  upon  the  presence  of  a  specific  ferment,  which  has 
been  termed  steapsin.  That  the  latter  view  is  probably  the  correct 
one,  appears  from  the  fact  that  the  same  result  is  obtained  if  the 
perfectly  fresh  gland  is  used  and  care  is  taken  to  prevent  the 
action  of  micro-organisms  by  adding  a  small  amount  of  hydro- 
cyanic acid  or  of  mercuric  chloride.  Of  the  nature  of  the  ferment 
nothing  is  known,  but  it  is  manifestly  very  unstable,  as  extracts 
prepared  from  glands  that  have  been  left  exposed^  to  the  air  for 
twenty-four  hours  are  perfectly  inert  when  brought  in  contact  with 
neutral  fats.  Apparently  it  is  digested  during  this  time  by  the 
trvpsin.  To  demonstrate  the  action  of  steapsin  on  fats,  a  small 
amount  of  perfectly  fresh  pancreas  is  finely  hashed  and  dehydrated 
with  90  per  cent,  alcohol.  It  is  then  dried  between  filter-paper  and 
placed  in  an  ethereal  solution  of  neutral  butyrin  (Butterfett).  On 
evaporation  of  the  ether  the  remaining  material  is  kept  between  two 
watch-crystals  at  a  temperature  of  from  37°  to  40°  C,  when  after 
awhile  a  distinct  odor  of  butyric  acid  becomes  manifest  (CI.  Bernard). 
Or,  a  small  amount  of  neutral  fat  is  treated  with  a  few  cubic  centim- 
eters of  a  feebly  alkaline  glycerin  extract  of  the  fresh  gland  (9 
parte  of  glycerin  and  1  part  of  al  per  cent,  solution  for  each  gramme 
of  the  gland),  to  which  a  few  drops  of  tincture  of  litmus  are  added. 
If  this  mixture  is  then  kept  at  a  temperature  of  37°  C,  it  will  be 
noted  that  the  alkalinity  gradually  decreases,  and  the  reaction  finally 
becomes  acid,  owing  to  the  liberation  of  free  fatty  acids  (Hammar- 
sten). 

The  emulsifying  action  of  the  pancreatic  juice  is  owing  to  the 
decomposition  of  the  neutral  fats  and  the  subsequent  saponification 
of  the  resulting  acids  by  the  alkaline  salts  which  are  at  the  same 
time  present. 

Maltase. — The  presence  of  maltase  in  the  pancreatic  juice  can 
only  be  inferred  indirectly  from  the  fact  that  small  amounts  of 
glucose  are  invariably  formed  during  the  action  of  the  aqueous 
nr  glycerin   extract  of  the  gland   upon   starch. 

Chymosin. — Chymosin  can  be  demonstrated  by  adding  a  small 
amount  of  the  pancreatic  juice  of  the  ox,  pig,  or  sheep  to  milk,  when 
coagulation  results,  as  hi  the  case  of  the  chymosin  of  the  gastric 
juice.  The  ferment,  however,  is  no!  present  in  all  mammals;  in 
dogs,  for  example,  it  i-  absent. 

The  glucolytic  ferment  of  Lepine  has  thus  far  not  been  isolated 
either  from  the  pancreatic  juice  or  from  the  gland  itself. 

I,,  conclusion,  it  is  to  !»<'■  noted  that  in  some  animals,  Buch  as  the 
rabbit,::  secretion  analogous  to  that  of  the  pancreas  is  furnished  also 
by  the  glands  of  Brunner,  which  are  found  in  the  upper  portion  <>f 
the  duodenum.  In  other  animals,  however,  such  as  the  dog,  the 
function  of  these  glands  i-  to  be  compared  to  that  of  the  pyloric 
glands  of  the  stomach  ;  and  according  to  Grutzner,  extract-  of  this 


140  THE  DIGESTIVE  FLUIDS. 

portion  of  the  mucous  membrane  contain  pepsin  in  considerable 
amount.  Whether  or  not  a  diastatic  ferment  also  is  here  secreted 
is  as  yet  undecided,  as  the  secretion  can  scarcely  be  obtained  uncon- 
taminated  by  pancreatic  juice,  and  it  is  hence  difficult  to  make 
definite  statements  regarding  its  composition. 

THE  ENTERIC  JUICE. 

The  enteric  juice  is  essentially  the  secretory  product  of  the  glands 
of  Lieberkiihu,  which  are  found  imbedded  in  the  mucous  membrane 
of  the  small  intestine,  and  to  some  extent  also  in  the  large  intestine. 
It  may  be  procured  by  resecting  a  loop  of  the  intestine,  measuring 
about  0.15-0.20  meter  in  length,  and  closing  the  proximal  end, 
while  the  distal  end  is  sutured  to  the  abdominal  wall.  The  mes- 
entery, however,  is  allowed  to  remain,  so  that  the  nerve-  and  blood- 
supply  of  the  portion  which  has  thus  been  isolated  is  interfered  with 
as  little  as  possible.  Instead  of  thus  forming  a  blind  pouch,  as  was 
first  done  by  Thiry,  both  ends  of  the  resected  loop  may  also  be 
sutured  to  the  anterior  abdominal  wall.  Such  fistulse  were  first 
established  by  Vella,  and  they  accordingly  bear  his  name.  The  free 
ends  of  the  divided  gut  are  in  either  case  then  united  with  each 
other  and  the  abdominal  wound  closed. 

In  animals  which  have  thus  been  operated  it  is  noted  that  after 
five  hours  following  the  ingestion  of  food  a  copious  secretion  of 
fluid  takes  place  in  the  small  intestine,  which  continues  for  about 
six  hours.  During  this  period  of  activity  the  mucous  membrane 
presents  a  rose-red  color,  while  it  is  pale  when  at  rest.  As  in  the 
case  of  the  pancreas,  the  secretion  is  intermittent.  It  can  be  mani- 
festly excited  in  a  reflex  manner,  as  a  moderate  secretion  may  be 
observed  within  an  hour  after  the  ingestion  of  food — that  is,  at  a 
time  when  but  little  chyme  has  passed  into  the  small  intestine. 
Later,  when  the  food  has  passed  the  stomach,  its  presence  alone  or 
that  of  the  digestive  products  which  have  been  formed  already, 
apparently  excites  the  increased  secretion  which  is  then  observed. 
This  may  be  further  increased  artificially  by  mechanical  and  espe- 
cially by  electrical  stimulation,  and  it  is  indeed  possible  to  cause  the 
secretion  of  enteric  juice  in  this  manner  even  at  a  time  when  diges- 
tion is  not  going  on. 

In  the  upper  portion  of  the  duodenum  of  the  dog  the  secretion  is 
said  to  be  small  in  amount,  mucoid,  and  jelly-like,  while  further  on 
it  becomes  more  fluid. 

As  obtained,  the  enteric  juice  always  contains  a  not  inconsiderable 
amount  of  mucus,  which  is  derived  from  the  goblet-cells  that  are 
found  along  the  entire  length  of  the  intestinal  canal.  The  small 
amorphous  flakes  which  are  always  found  in  the  secretion  consist 
entirely  of  mucus.  The  juice  itself,  which  can  be  separated  from 
the  greater  portion  of  the  mucus  by  filtration,  is  in  the  lower  por- 
tion of  the  small  intestine  a  thin  light-yellowish  fluid,  of  a  strongly 


THE  BILE.  1-11 

alkaline  reaction.  This  is  largely  due  to  the  presence  of  consider- 
able amounts  of  sodium  carbonate,  and  we  accordingly  find  that  on 
the  addition  of  an  acid  effervescence  of  carbon  dioxide  occurs.  Its 
specific  gravity  in  the  dog  is  fairly  constant,  and  corresponds  to 
about  1.01 0-1.011. 

The  amount  of  solids  is  largely  dependent  upon  the  character  and 
the  quantity  of  the  food  ingested,  and  in  the  dog  may  vary  between 
12.2  and  24.1  pro  mille.  These  variations  are  mainly  referable  to 
the  presence  of  albumins,  which  are  always  found  in  the  enteric 
juice,  while  the  inorganic  constituents  are  fairly  constant.  A 
general  idea  of  the  chemical  composition  of  the  secretion  may  be 
formed  from  the  accompanying  analyses  : 

Dog  Horse 

(Thiry).  (Colin). 

"Water  97.59  per  cent.         98.10  per  cent. 

Solids 2.41    «      «  1.90  "        " 

Albumins 0.80  "1         o  45   "        " 

Other  organic  matter  (mucin)  .  0.73   "      "     / 

Mineral  asli 0.88   "      "  1.45  "        " 

Sodium  carbonate    ....  0.40  " 

Sodium  chloride 0.48  "      " 

Of  the  amount  of  enteric  juice  which  is  secreted  under  normal 
conditions  in  twenty-four  hours  we  know  nothing.  In  disease,  how- 
ever, and  notably  in  Asiatic  cholera  exceedingly  large  quantities 
may  be  observed  ;  but  it  is  probable  that  in  such  cases  we  are  deal- 
ing with  a  direct  transudation  from  the  blood,  and  not  with  an 
actual  secretory  product  of  the  cells.  On  section  of  the  correspond- 
ing nerves  hypersecretion  can  be  artificially  brought  about.  This 
may  be  compared  to  the  paralytic  saliva  which  is  obtained  from 
the' sublingual  gland  on  section  of  the  chorda  and  of  the  sympathetic 
fibres  that  supply  the  gland. 

The  question  whether  or  not  the  enteric  juice  plays  a  part  in  the 
process  of  digestion  is  still  undecided.  This  uncertainty  is  largely 
owing  to  the  fact  that  innumerable  bacteria  are  always  present  in 
the  enteric  secretion,  and  that  some  of  these  possess  the  power  of 
inverting  starch  and  of  digesting  albumins.  Schiff,  on  the  other 
hand,  claims  that  these  digestive  phenomena  are  directly  referable 
to  the  action  of  ferments,  which  are  secreted  by  the  glands  of 
Lieberkuhn.  However  this  may  be,  it  appears  certain  that  the 
enteric  juice  contains  ferments  which  are  capable  of  inverting 
saccharose  and  maltose,  and  that  these  ferments  are  furnished  by  the 
glands  in  question. 

Like  the  pancreatic  juice,  the  enteric  secretion  is  capable  of  emul- 
sifying fats,  bul  it  is  doubtful  whether  it  can  also  bring  about  their 
saponification. 

THE  BILE. 

Formerly  it  was  generally  supposed  thai  the  bile  played  an  im- 
portant part  in  the  process  of  digestion,  and  was  further  capable  of 


142  THE  DIGESTIVE  FLUIDS. 

controlling  the  intensity  of  the  putrefactive  and  fermentative  proc- 
esses which  even  normally  take  place  in  the  lower  intestinal  tract. 
It  has  now  been  definitely  established,  however,  that,  aside  from  its 
emulsifying  action  upon  fats,  the  secretion  possesses  no  digestive 
properties  whatever,  and  is  likewise  without  effect  upon  the  bacteria 
which  are  normally  found  in  the  intestinal  canal.  We  accordingly 
find  that  in  animals  and  in  man  the  processes  of  nutrition  are  in  no 
way  interfered  with  if  the  bile  is  prevented  from  entering  the 
digestive  tube,  but  is  carried  to  the  outside  through  the  establish- 
ment of  a  fistulous  opening  in  the  common  duct,  provided  that  food 
is  administered  which  contains  but  little  fat.  With  a  diet  consist- 
ing of  albumins  and  carbohydrates  digestion  thus  continues  unim- 
paired, and  the  animal  is  capable  of  maintaining  its  nitrogenous 
equilibrium  practically  as  before.  If  fats,  however,  are  given  at  the 
same  time  in  large  amounts,  more  or  less  serious  digestive  disturb- 
ances soon  develop,  and  the  animal  loses  weight.  In  such  cases  it 
has  been  ascertained  that  whereas  normally  from  2  to  10  per  cent, 
of  the  ingested  fat  is  eliminated  in  the  feces,  from  31  to  47  per  cent, 
now  escapes  resorption.  The  offensive  gases  which  are  then  passed 
by  the  animal  are  referable  to  an  increase  of  the  putrefactive  proc- 
esses in  the  intestines,  it  is  true.  This  is,  however,  not  owing  to 
the  absence  of  bile  per  se,  but  to  the  fact  that  the  unabsorbed  fats 
envelop  the  albuminous  material,  and  thus  prevent  its  further  diges- 
tion, so  that  in  the  lower  portion  of  the  digestive  canal,  where  the 
putrefactive  processes  are  most  intense,  the  bacteria  find  an  increased 
amount  of  pabulum  at  their  disposal,  and  an  increase  of  the  putre- 
factive products  accordingly  results.  In  the  presence  of  bile,  on  the 
other  hand,  this  does  not  occur,  as  its  emulsifying  effect  upon  the 
fats  soon  leads  to  their  absorption,  and  thus  leaves  the  albumins 
exposed  to  the  action  of  the  digestive  juices,  and  to  their  resorption 
in  turn.  Indirectly,  it  can  thus  control  the  process  of  putrefaction, 
but  such  action  is  not  due  to  any  germicidal  or  antiseptic  properties 
of  its  own.  On  withdrawing  fats  from  the  food  and  giving  an  ade- 
quate supply  of  carbohydrates,  normal  relations  are  soon  re-estab- 
lished, though  the  bile  is  absent  as  before. 

The  bile  in  reality  represents  a  most  important  excretory  product 
of  the  animal  body,  and  may  in  this  sense  be  compared  to  the 
urine.  It  appears,  moreover,  that  those  waste-products  which  are 
markedly  toxic  in  action,  and  could  not  be  carried  to  the  kidneys 
through  the  blood-current  without  seriously  disturbing  the  general 
health,  are  formed  in  the  liver  directly,  and  are  hence  removed 
through  separate  channels  in  the  bile. 

Substances  are  further  eliminated  in  this  manner  which,  like 
cholesterin,  are  insoluble  in  water,  and  could  hence  not  be  excreted 
by  the  kidneys. 

For  convenience'  sake,  however,  the  bile  is  described  at  this  place. 

Secretion. — As  found  in  the  gall-bladder,  the  bile  represents  the 
secretory  product  of  the  liver-cells  which  is  eliminated    into    the 


THE  BILE.  143 

radicles  of  the  biliary  passages,  together  with  so-called  mucus  which 
is  derived  from  the  epithelial  lining  of  the  greater  trunks  and  the 
gall-bladder  itself.  Its  secretion  is  continuous,  but  liable  to  exacer- 
bations which  are  essentially  dependent  upon  the  ingestion  of  food. 
According  to  Heidenhain,  the  curve  of  secretion,  in  reference  to 
amount,  shows  two  periods  of  greatest  activity,  which  in  the  dog 
corre.-jxmd  to  the  third  to  the  fifth  and  the  thirteenth  to  the  fifteenth 
hour,  respectively,  after  the  administration  of  food.  This  curve, 
however,  is  further  influenced  by  the  character  of  the  food  ingested. 
With  an  albuminous'  diet  three  stages  of  maximal  secretion  are 
thus  noted  :  The  first  after  two  to  three  hours,  a  second  stage 
after  five  to  eight  hours,  and  a  third  stage  after  twelve  to  fourteen 
hours.  With  a  diet  of  albumins  and  fits,  on  the  other  hand,  the 
period  of  greatest  secretion  occurs  after  eleven  to  twelve  hours, 
and  with  one  of  albumin  and  carbohydrates  after  from  nine  to 
fourteen  hours.  These  figures  have  reference  to  observations  which 
were  made  after  the  administration  of  only  one  meal  in  the  twenty- 
four  hours.  With  two  meals  analogous  results  are  obtained.  The 
individual  periods,  however,  are  shorter;  and  as  the  number  of 
feedings  is  increased  the  secretion  becomes  more  uniform,  so  that 
with  a  meal  every  two  hours  no  variations  of  moment  can  be 
discerned. 

Amount. — The  amount  of  bile  which  is  eliminated  in  the  twenty- 
four  hours  is  variable  even  under  normal  conditions.  In  dogs  from 
2.9  to  36.4  grammes  can  be  obtained  pro  kilogramme  of  weight  of 
the  animal.  In  man  the  secretion  apparently  varies  between  400 
and  800  grammes ;  but  it  is  possible  that  these  figures  do  not  repre- 
sent normal  values.  Ranke  has  estimated  that  a  man  weighing  75 
kilogrammes  secretes  about  1050  grammes  even  in  health.  To  a 
certain  extent  the  amount  is  influenced  by  the  character  of  the  food, 
and  it  appears  to  be  quite  generally  accepted  that  a  diet  rich  in 
albumins  will  call  forth  a  greater  secretion  of  bile,  while  the  carbo- 
hydrates are  though!  to  diminish  its  amount,  or  are  at  least  incapable 
of  increasing  this,  like  the  albumins.  The  fats,  on  the  other  hand, 
are  probably  without  effect  in  either  direction. 

It  was  formerly  thought  that  a  number  of  drugs  could  increase 
the  How  of  the' bile,  and  physicians  were  wont  to  administer 
cholagogues  when  they  supposed  that  the  secretion  of  bile  was 
deficient.  This  view  has  now  been  abandoned,  as  it  has  been  defi- 
nitely established  that  drugs  are  without  effect  in  this  direction. 
The  only  cholagogue,  indeed,  if  it  may  so  be  termed,  is  the  bile 
itself.  This  is  readily  understood,  if  we  bear  in  mind  that  the 
bile  i-  essentially  an  excretory  product. 

General  Properties.— The  color  of  the  bile  differs  m  different 
animals,  and  may  vary  from  a  bright  yellow  to  an  intense  grass- 
green,  with  various  shades  of  brown  and  blue,  [n  man  it  is  usually 
of  a  golden-yellow  color,  but  it  may  at  time-  appear  bright  green 
even  when  perfectly  fresh. 


144  THE  DIGESTIVE  FLUIDS. 

While  pure  bile,  uncontaminated  with  mucus,  is  a  thin  transparent 
fluid,  that  which  is  eliminated  into  the  intestinal  tract  and  is  found 
in  the  gall-bladder  is  markedly  viscid  and  more  or  less  turbid.  Its 
taste  is  intensely  bitter,  with  a  sweetish  and  nauseating  after-taste. 
The  odor  is  in  many  animals  peculiar,  and  at  times  suggestive  of 
musk.  The  reaction  is  normally  alkaline.  After  exposure  to  the 
air,  however,  it  soon  becomes  acid,  but  becomes  alkaline  again  owing 
to  the  development  of  trimethylamin  and  ammonia.  The  specific 
gravity  varies  between  1.010  and  1.033.  In  the  gall-bladder  it  is 
higher  (1.026-1.033),  where  a  constant  resorption  of  water  is  going 
on,  and  where  considerable  amounts  of  mucus  are  added  to  the  bile, 
than  in  the  bile  proper,  which  is  secreted  by  the  liver  (1.010-1.012). 
We  accordingly  find  that  the  amount  of  solids  is  greater  in  the 
bladder-bile  than  in  that  which  may  be  termed   the  hepatic  bile. 

Chemical  Composition. — The  following  analyses  show  the  gen- 
eral chemical  composition  of  the  bile  in  different  animals,  and  also 
illustrate  the  differences  which  exist  between  bladder-bile  and 
hepatic  bile : 

Human  bladder-bile       Human     hepatic     bile    (normal), 
(normal).  (Frerichs.)  (Hammarsten,  1  to  3;  Zeynek,  4.) 


1.               '  2.                     1.                   2.  3.  4. 

Water      860.0         859.2         974.80       964.74  974.60  989.24 

Solids 140.0        140.8          25.20        35.26  25.40  30.76 

JJucm \       26.6          29.8            5.29          4.29  5.15  2.08 

-Pigments     ....   J 

Salts  of  bile  acids  .    .        72.2           91.4            9.31         18.24  9.04  18.31 

Taurocholates 3.03           2.17  2.18 

Glycocholates 6.27         16.16  6.86  .    . 

Fatty  acids  (soaps)     .         .    :            .    .              1.23           1.36  1.01  2.08 

Cholesterin      ....          1.6             2.6             0.63           1.60  1.50  2.30 J 

Lecithin \         ft  99        /  0.57  0.65  0.73 

Fat 3.2            9.2/         u,zz        \  0.95  0.61  .    . 

Soluble  salts    -   .    .  \         .  ,            77      J      8.07           6.76  7.25  9.10 

Insoluble  salts     .    .   /         °'°                '      \     0.2o          0.49  0.21  0.81 

Analyses  of  the  Beadder-bile  of  Animaes. 

Dog.                   Pig.                  Ox.  Birds.  Shad. 

(Hoppe-Seyler.)  (Grundelach-  (Berzelius.)  (Marsson.)  (Schloss- 

Strecker.)  berger.) 

Water 813.56             888.0            904.4  800.2  944.8 

Solids 186.44            112.0              95.6  199.8  55.2 

1  170.6  36.3 


Sodium  glycocholate  •    •    \  000 

Sodium  taurocholate        122.8    f  M'° 


Cholesterin      ....  2.91") 

Fats       15.11  I 

Soaps 16.03  ( 

Lecithin 18.11  J 


22.3 


80.0  I 

}■  3.6 

J 


Mucin       3.49  5.9  \  f  25.6               14.8 

Other  organic  solids;  \  „  r.  Y  3.0           ]  '    ' 

insoluble  in  alcohol  I  J  I 

Inorganic  solids  ...  .    .  .    .  12.6  21.0                •    • 

Analyses  of  the  inorganic  salts  have  given  the  following  results, 
which  are  taken  from  Jacobsen  and  Hoppe-Seyler,  respectively. 
The  figures  have  reference  to  100  parts  by  weight  of  mineral  ash : 

>  Including  fat. 


THE  BILE. 


145 


Man 
(hepatic  bile). 

Sodium  chloride 65.16 

Potassium   chloride 3.39 

Sodium  carbonate 11.16 

Trisodium  phosphate 15.90 

Tricalcium  phosphate        4.44 

Calcium  carbonate      

Potassium  sulphate variable 

Sodium  sulphate .    . 

Iron,  Silica      1  4 

Ar     '     .                                                           >  traces 

Magnesium,  copper J 


Ox 

(bladder-bile). 
7.50 l 

2.50 

40.0 

9.50 

2.0 

25.0 

traces 


The  Mucinous  Body  of  the  Biles. 

The  mucinous  body  which  is  found  in  the  bladder-bile  of  all 
animals  is  apparently  of  a  different  nature  in  different  animals.  The 
mucus  of  the  human  bile  thus  largely  consists  of  true  mucin,  while 
in  ox-bile  a  mucinous  nucleo-albumin  is  principally  found.  To 
isolate  this  latter,  the  bile  is  precipitated  with  5  times  its  volume 
of  absolute  alcohol  and  immediately  centrifugalized.  The  super- 
natant liquid  is  poured  off,  and  the  sediment  rapidly  dried  with 
filter-paper  and  dissolved  in  water.  By  repeating  this  process  the 
substance  can  be  obtained  in  a  fairly  pure  form.  Its  character  as 
a  nucleo-albumin  becomes  apparent  on  treating  its  neutral  solutions 
with  a  small  amount  of  hydrochloric  acid.  A  flocculent  precipitate 
then  develops,  which  readily  dissolves  upon  the  further  addition  of 
hydrochloric  acid  to  the  extent  of  0.3  per  cent.  This  solution  re- 
mains clear  for  a  long  time  even  when  kept  at  the  temperature  of 
the  body.  If  a  small  amount  of  pepsin,  however,  is  added,  a  sepa- 
ration of  nuclein  occurs.  On  fusing  the  dried  substance,  moreover, 
with  potassium  hydrate  and  sodium  nitrate,  an  amount  of  phosphoric 
acid  is  obtained  which  is  greatly  in  excess  of  the  amount  required 
to  saturate  all  of  the  mineral  ash  that  is  present  when  calculated  as 
tricalcium  phosphate.  On  boiling  with  a  dilute  mineral  acid  no 
reducing-substance  is  formed,  as  in  the  case  of  true  mucin.  Acetic 
acid  precipitates  the  substance  from  its  solutions,  in  the  absence  of 
biliary  acids  ;  but  this  precipitate,  unlike  that  of  mucin,  is  soluble 
in  an  excess  of  the  acid. 

The  mucin  proper  which  is  found  in  human  bile  may  be  isolated, 
as  already  described  (page  113). 


The   Biliary  Acids. 

The   biliary  acids  which    are  normally  found  only  in  the  bile  arc 

essentially  compound  amido-acids,  which  arc  formed  through   the 

union  of  glycocoll  on  the  one  hand,  and  tanrin  on   the  Other,  with  a 

cholalic  acid.     In  the  bile  of  sharks  Elammarsten  discovered  the  ex- 

istence  of  a  third  group  of  biliary  acids,  which  are  rich  in  sulphur, 

and,  like  the    conjugate   sulphates  of  the  urine,  yield    sulphuric  acid 

'  Tbi-  figure  '-  too  low,  owing  to  the  farl  thai  Hoppe  Seyler'i  analysis  lias  reference  t'>  i be 
Inorganic  salts,  which  werenol  dissolved  by  alcohol. 

10 


146  THE  DIGESTIVE  FLUIDS. 

on  boiling  with  hydrochloric  acid.  Traces  of  these  acids  are  said 
to  occur  also  in  human  bile.  Of  their  chemical  nature,  however, 
nothing  is  known. 

Under  pathologic  conditions,  when  the  free  outflow  of  bile  is 
impeded,  owing  to  a  swelling  of  the  mucous  membrane  of  the 
common  duct  or  to  the  presence  of  a  calculus,  resorption  of  the 
bile  takes  place  through  the  lymph-channels,  and  the  secretion 
thus  finds  its  way  into  the  blood.  Jaundice  then  results,  and  in 
such  cases  not  only  the  bile-pigments,  but  also  the  bile-acids  can 
be  demonstrated  in  the  general  circulation.  To  the  presence  of  the 
latter  is  due  the  slow  pulse  which  is  so  constantly  observed  in 
icterus.  At  the  same  time  the  bile-acids  bring  about  a  dissolution 
of  the  red  corpuscles,  and  thus  manifest  their  true  nature  as  excretory 
products. 

Regarding  the  origin  of  the  biliary  acids,  we  know  only  that 
they  are  formed  exclusively  in  the  liver,  but  of  the  manner  in 
which  their  formation  takes  place  we  are  ignorant. 

The  amido-radicles  of  the  bile-acids  are  unquestionably  formed  in 
the  general  nitrogenous  metabolism  of  the  body,  and,  as  such,  are 
found  in  other  organs  besides  the  liver.  Glycocoll  is  then  to  a  cer- 
tain extent  further  oxidized,  and  contributes  to  the  formation  of 
urea.  Of  the  ultimate  fate  of  taurin,  we  know  that  its  sulphur  can 
be  oxidized  to  sulphuric  acid  and  be  eliminated  in  the  urine  in  this 
form.  In  the  human  being  traces  are  further  excreted  as  tauro- 
carbaminic  acid  ;  and  in  rabbits,  at  least,  its  hypodermic  injection 
leads  to  the  elimination  of  the  body  as  such.  It  is  thus  difficult  to 
understand  why  two  substances  like  these,  for  the  removal  of  which 
the  animal  body  has  manifestly  other  means  at  its  disposal  than 
their  elimination  through  the  bile,  should  here  appear.  We  may 
imagine,  however,  that  the  formation  of  the  bile-acids  in  the  liver 
represents  a  conservation  of  energy  on  the  part  of  the  body,  and 
further  constitutes  a  reserve  mechanism  by  which  Avaste-products 
can  be  removed  in  a  state  of  incomplete  oxidation. 

Of  the  origin  of  the  non-nitrogenous  acids,  which  combine  with 
glycocoll  and  taurin  to  form  the  biliary  acids,  we  know  nothing. 
The  synthesis,  however,  manifestly  occurs  in  the  liver,  and  here 
only,  as  after  extirpation  of  the  organ  in  birds  and  frogs  bile-acids 
are  not  found  in  the  blood.  In  mammals,  also,  neither  bile- 
pigments  nor  bile-acids  can  here  be  demonstrated  after  ligating 
the  gall-duct  and  the  thoracic  duct. 

In  the  bile  of  most  animals  the  biliary  acids  occur  in  combination 
with  sodium,  while  in  sea-fish  they  are,  curiously  enough,  present  as 
potassium  salts. 

As  regards  the  relative  quantity  of  the  two  principal  biliary  acids 
which  are  found  in  the  bile,  it  is  to  be  noted  that  in  man  glycocholic 
acid  is  usually  more  abundant  than  taurocholic  acid.  In  strictly 
carnivorous  animals,  on  the  other  hand,  the  latter  only  is  found  ; 
but  the  same  also  holds  good  for  certain  herbivora,  such  as  the  sheep 


THE  BILE.  147 

and  the  goat.  In  other  animals  the  relation  is  variable,  and  in 
some,  it  is  stated,  glycocholic  acid  only  is  found. 

Isolation. — Collectively,  the  biliary  aeids,  in  the  form  of  their 
salts,  can  be  obtained  in  the  following  manner  :  the  bile  is  mixed 
with  animal  charcoal,  evaporated  to  dryness,  and  the  residue  ex- 
tracted with  absolute  alcohol.  This  extract,  which  contains  the 
biliary  acid  salts,  cholesterin,  fats,  soaps,  and  lecithin,  is  then 
filtered,  concentrated,  and  treated  with  a  large  excess  of  ether.  In 
this  manner  the  salts  of  the  bile-acids  are  precipitated,  while  the 
other  substances  remain  in  solution.  On  standing,  the  precipitate 
gradually  becomes  crystalline,  and  is  then  spoken  of  as  Planner's 
crystallized  bile.  In  this  form  the  biliary  acids  are  conveniently 
estimated  as  a  whole.  If  then  it  is  desired  to  determine  the  rela- 
tive amount  of  the  two  principal  acids  present,  it  is  only  necessary 
to  estimate  the  sulphur  in  Platner's  bile,  from  which  the  corre- 
sponding amount  of  taurocholic  acid  can  be  calculated. 

To  separate  the  two  acids  from  each  other,  Platner's  bile  is  dis- 
solved in  water  and  completely  precipitated  with  a  solution  of  neutral 
acetate  of  lead.  The  corresponding  lead  salts  are  thus  formed,  and 
■can  now  be  readily  separated  from  each  other,  as  the  glycocholate  is 
thrown  down,  while  the  taurocholate  remains  in  solution.  In  the 
filtrate  the  latter  is  then  precipitated  with  ammonia.  To  obtain  the 
free  acid  from  the  glycocholate,  the  precipitated  lead  salt  is  sus- 
pended in  water  and  evaporated  to  dryness  in  the  presence  of  sodium 
carbonate.  The  sodium  salt  is  thus  obtained  and  extracted  with 
alcohol.  The  alcohol  is  evaporated  off,  the  residue  dissolved  in 
water  and  treated  with  hydrochloric  acid,  when  the  glycocholic  acid 
will  separate  out.  The  taurocholic  acid  can  be  similarly  obtained 
from  its  lead  salt  by  decomposing  the  solution  with  hydrogen  sul- 
phide. The  resulting  plumbic  sulphide  is  filtered  off,  the  filtrate 
evaporated  to  dryness,  the  residue  dissolved  with  a  small  amount 
of  alcohol,  and  then  precipitated  with  an  excess  of  ether,  when  the 
free  acid  is  thrown  down. 

Tests  for  the  Bile-acids. — Pettenkofer's  Test. — On  treating  the 
biliary  acids  in  aqueous  or  alcoholic  solution  with  a  few  drops  of  an 
0.1  per  cent,  solution  of  furfurol  and  1  or  2  c.c.  of  concentrated, 
chemically  pure  sulphuric  acid,  a  beautiful  purple  color  develops. 
AjB  furfurol  results  when  concentrated  sulphuric  acid  is  added  to 
a  carbohydrate,  the  test  may  also  be  conducted  by  treating  the  solu- 
tion of  the  biliary  acids  (1  or  2  c.c)  with  a  few  drops  of  a  10  per 
cent,  solution  of  cane-SUgar  and  then  with  sulphuric  acid.  In  either 
case,  however,  care  should  be  hail  that  the  temperature  which  results 

during  the-  reaction  does  not  exceed  70°  ( '.,  as  otherwise  the  result- 
ing pigment  i-  destroyed.  This  may  be  obviated  by  substituting 
-tron_'  phosphoric  acid  for  the  sulphuric  acid,  and  placing  the  solu- 
tion in  boiling  water.  The  resulting  pigment  shows  two  bands  of 
absorption  on  spectroscopic  examination.     One  of  these  is  situated 

at    F;    the    other    between    I)    and     E,    near    E.      On    diluting   with 


148  THE  DIGESTIVE  FLUIDS. 

alcohol  a  green  fluorescence  is  observed.  In  the  presence  of  an 
excess  the  red  pigment  disappears,  but  reappears  upon  the  further 
addition  of  sulphuric  acid.  On  standing,  the  color  gradually  turns 
to  a  bluish  violet. 

As  Pettenkofer's  reaction  can  also  be  obtained  with  other  sub- 
stances, such  as  the  phenols,  the  higher  alcohols,  certain  bases  of 
the  aromatic  series,  the  higher  hydrocarbons,  and  acids  of  the  fatty 
series  and  the  benzol  series,  it  is  always  necessary  to  isolate  previ- 
ously the  biliary  acids  as  Platner's  bile,  before  drawing  conclusions 
from  the  reaction.  Albumins,  if  present,  must  in  every  case  first 
be  removed.  For,  as  has  been  stated,  many  of  these  contain  carbo- 
hydrate groups,  which  on  contact  with  concentrated  sulphuric  acid 
give  rise  to  the  formation  of  furfurol.  This  in  turn  combines  with 
the  aromatic  oxy-acids  and  phenols  that  result  from  decomposition 
of  the  amido-acids  to  form  colored  compounds,  which  are  either 
identical  with  or  very  similar  to  those  obtained  in  the  case  of  the 
biliary  acids  (^see  the  reaction  of  Adamkiewicz  and  the  hydrochloric 
acid  test  for  albumins). 

Pettenkofer's  reaction  is,  in  the  case  of  the  biliary  acids,  referable 
to  the  non-nitrogenous  acid  constituents  of  these  acids,  viz.,  to 
cholalic  acid  or  one  of  its  congeners. 

Physiological  Test. — Aside  from  their  reaction  with  furfurol,  the 
bile-acids,  or  their  salts,  may  also  be  identified  as  such  from  their 
effect  upon  the  action  of  the  heart.  To  this  end,  the  heart  of  a  cura- 
rized  frog  is  exposed  and  moistened  with  a  small  drop  of  a  1  per 
cent,  solution  of  atropin,  so  as  to  eliminate  the  action  of  the  vagus. 
A  few  drops  of  an  aqueous  solution  of  the  bile-acids  are  then 
placed  upon  the  heart,  when  their  retarding  action  can  readily  be 
demonstrated. 

Glycocholic  Acid. — As  has  been  stated,  glycocholic  acid  is  the 
principal  biliary  acid  that  is  found  in  the  bile  of  man.  It  is  formed 
through  the  union  of  glycocoll  and  cholalic  acid,  as  represented  by 
the  equation  : 

C2H5N02  +  C2.H40O5  =  C26H43N06  +  H20 

Glycocoll.         Cholalic        Glycocholic 
acid.  acid. 

It  is  accordingly  decomposed  into  these  constituents  when  treated 
with  dilute  acids  or  alkalies  under  the  application  of  heat. 

In  the  pure  state  glycocholic  acid  occurs  in  the  form  of  fine,  silk- 
like needles,  which  are  readily  soluble  in  alcohol,  less  readily  so  in 
water,  and  insoluble  in  ether.  From  its  aqueous  solutions  it  is 
readily  precipitated  on  adding  a  small  amount  of  a  mineral  acid. 
The  crystals  melt  at  133°  C.  It  is  a  monobasic  acid,  and  forms 
salts  which,  with  the  exception  of  the  lead  and  silver  compounds, 
are  all  soluble  in  water  and  alcohol.  The  free  acid  and  its  salts 
give  Pettenkofer's  reaction,  and  turn  the  plane  of  polarization  to  the 
right. 

On  heating  glycocholic  acid  with  concentrated  sulphuric  acid  the 


THE  BILE.  149 

anhvdride  of  glycocholic  acid  is  precipitated  in  the  form  of  oily- 
droplets,  which  subsequently  tend  to  coalesce.  This  anhydride  is 
termed  cholonic  acid,  and  has  the  composition  C20H41NO5. 

According  to  Michailoff,  glycocholic  acid  when  treated  with  con- 
centrated sulphuric  acid  in  the  presence  of  acetic  acid  is  said  to  yield 
an  orange  color  with  a  green  fluorescence.  On  salting  with  ammo- 
nium sulphate  a  precipitate  is  formed  which  in  its  reactions  is 
identical  with  biliverdin.  Urobilin  is  said  to  remain  in  solution. 
This  observation  is  of  special  interest,  as  showing  the  possible  rela- 
tionship which  may  exist  between  the  biliary  acids  and  the  bile-, 
pigments.  We  find,  as  a  matter  of  fact,  that  an  increase  in  the 
production  of  bile-pigments  on  the  part  of  the  liver  is  associated 
with  a  diminished  formation  of  biliary  acids.  Others  have  con- 
cluded from  this  observation  that  a  connection  between  the  produc- 
tion of  bile-acids  and  bile-pigments  does  not  exist,  and  that  the 
origin  of  the  two  classes  of  substances  must  be  referred  to  a  separate 
activity  on  the  part  of  the  hepatic  cells.  It  appears  to  me,  however, 
that  this  inference  is  not  altogether  justifiable. 

Closely  related  to  the  common  glycocholic  acid  is  the  so-called  hyo- 
glycocholic  acid,  which  has  been  found  in  small  amounts  in  the  bile 
of  the  pig.  On  decomposition  it  yields  glycocoll  and  hyocholalic 
acid,  as  shown  in  the  equation  : 

C27H43X05    +    H20    =    C2H5N02    +    C25H40O4 
Hyoglycocholic  Glycocoll.  Hyocholalic 

acid.  acid. 

The  substance  itself  is  almost  insoluble  in  water,  but  soluble  in 
alcohol.  It  is  crystallizable,  but  usually  obtained  as  a  resinous 
mass.  Its  salts  are  precipitated  from  their  solutions  by  calcium 
chloride,  barium  chloride,  and  magnesium  chloride.  By  salting 
with  sodium  sulphate  they  separate  out  like  soaps.  Like  the  com- 
mon glycocholates,  they  give  Pettenkofer's  reaction. 

In  addition  to  these  two  forms  still  other  glycocholates  apparently 
exist.  In  the  bile  of  rodents  a  glycocholate  is  thus  found,  which 
cannot  be  salted  out  with  sodium  sulphate,  but  which  is  also  precipi- 
tated by  the  salts  of  the  alkaline  earths.  Of  its  nature,  however,  as 
also  of  the  so-called  guano-bUiary  acid3  which  apparently  belongs  to 
this  order,  nothing  is  known. 

Isolation. — The  common  glycocholic  acid  is  most  conveniently 
obtained  by  starting  with  Platner's  bile,  that  has  been  prepared  from 
human  bile  or  from  that  of  the  ox,  as  already  described. 

Hyoglycocholic  acid  fan  be  isolated  from  the  bile  of  the  pig  by 
fir-'  decolorizing  with  animal  charcoal  and  then  salting  with  sodium 
sulphate  in  substance.  The  acid  is  thus  precipitated,  ami  can  then 
he  filtered  off.  It  IB  washed  with  a  solution  of  the  salt,  dissolved  in 
water,  and    precipitated    in    the    form  of  the    free  acid    by    means  of 

hydrochloric  acid. 

Taurocholic  Acid. — TaurochollC  acid,  as  has    been    slated,  is   the 

only  biliary  acid  that  is  found  in   the  bile  of  the  purely  carnivor- 


150  THE  DIGESTIVE  FLUIDS. 

ous  animals,  but  it  also  occurs  in  the  bile  of  man  and  most  herbiv- 
orous animals.  Among  these,  the  sheep  and  goat  are  especially 
noteworthy,  as  in  these,  like  the  pure  carnivora,  taurocholic  acid 
only  is  found.  It  is  formed  synthetically  in  the  liver  from  taurin 
aud  cholalic  acid,  and  is  accordingly  resolved  into  its  components 
by  treating  with  dilute  acids  or  alkalies  under  the  application  of 
heat.  The  same  result  indeed  is  reached  by  evaporating  the  bile 
together  with  water  or  allowing  it  to  undergo  putrefaction.  The 
chemical  change  is  represented  by  the  equation  : 

C26H45NS07  +  H20  =  C2H7NS03  +  C24H40O5 
Taurocholic  Taurin.  Cholalic 

acid.  acid. 

In  the  pure  state  taurocholic  acid  occurs  in  the  form  of  fine  deli- 
quescent needles,  which  are  soluble  in  water  and  alcohol,  but 
insoluble  in  ether.  It  is  capable  of  maintaining  glycocholic  acid  in 
solution,  and  it  is  for  this  reason  that  the  latter  acid  cannot  be  pre- 
cipitated from  the  bile  by  adding  a  mineral  acid  when  taurocholic 
acid  is  at  the  same  time  present  in  sufficient  amount.  Like  glyco- 
cholic acid,  it  is  a  monobasic  acid,  and  forms  salts  which  for  the 
most  part  are  soluble  in  water  and  in  alcohol.  Its  salts  with  the 
alkalies  are  precipitated  from  their  solutions  by  subacetate  of  lead 
and  ammoniacal  subacetate  of  lead,  but  not  by  the  neutral  acetate,  by 
copper  sulphate,  or  silver  nitrate.  Curiously  enough,  the  sodium 
salt  obtained  from  the  bile  of  the  ox  is  dextrorotatory,  while  the 
same  salt  which  is  found  in  that  of  the  dog  turns  the  plane  of 
polarization  to  the  left.  An  isomerism  thus  apparently  exists  which 
is  analogous  to  that  observed  in  the  case  of  the  tartaric  acids. 

Taurocholic  acid  forms  emulsions  with  the  peptones,  but  does  not 
precipitate  them,  as  is  generally  stated  ;  but  it  does  precipitate  albu- 
mins, syntonins,  and  albumoses. 

Hyotaurocholic  acid  is  the  biliary  acid  which,  as  a  sodium  salt,  is 
found  in  the  bile  of  pigs,  and  is  analogous  to  the  hyoglycocholic  acid 
already  described.  Its  amount,  however,  is  small.  On  decomposi- 
tion it  yields  taurin  and  hyocholalic  acid,  as  shown  in  the  equation  : 

C26H45NSOfi  +   H20  =  C^EUO,   +   C2H7NS03 

Hyotaurocholic  Hyocholalic  Taurin. 

acid.  acid. 

Chenotaurocholic  acid  is  the  most  important  biliary  acid,  which 
is  found  in  the  bile  of  geese.  It  is  indistinctly  crystalline,  and  is 
soluble  in  water  and  alcohol.  Like  the  acids  already  described,  it 
gives  Pettenkofer's  reaction.  On  prolonged  boiling  with  alkalies 
it  is  decomposed  into  taurin  and  chenocholalic  acid,  as  shown  in  the 
equation  : 

C29H49NS06  +   H20  =  C27H4404   +   C2H7NS03 

Chenotaurocholic  Chenochola-  Taurin. 

acid.  lie  acid. 

Isolation. — Taurocholic  acid  is  most  conveniently  obtained  from 
Platner's  bile  of  man  or  the  ox,  as  already  described.     To  isolate 


THE  BILK  151 

it  from  the  bile  of  dogs,  the  fluid  is  shaken  with  animal  charcoal 
and  alcohol,  then  decanted  and  filtered.  The  filtrate  is  evaporated 
to  dryness,  the  residue  taken  up  with  a  small  amount  of  warm 
absolute  alcohol,  and  filtered.  The  clear  solution  is  then  treated 
with  ether  until  it  becomes  cloudy.  On  standing,  the  taurocholate 
of  -odium  separates  out  in  the  form  of  fine  crystals.  These  are 
dissolved  in  water  and  treated  with  ammoniacal  subacetate  of  lead. 
The  resulting  precipitate  is  suspended  in  alcohol  and  decomposed 
with  hydrogen  sulphide.  The  free  acid  i-  thus  liberated,  and  can 
he  obtained  as  such  by  evaporating  the  filtrate  to  dryness  or  by 
diluting  with  ether. 

To  isolate  chenotaurocholic  acid,  the  bile  of  geese  is  first  treated 
with  strong  alcohol,  to  remove  the  mucus.  The  filtrate  is  mixed 
with  ether  and  set  aside,  when  the  biliary  salts  separate  out  as 
a  glutinous  mass,  which  is  then  washed,  dried,  redissolved  in  99  per 
cent,  alcohol,  and  again  precipitated  with  ether.  The  crystalline 
deposit  which  is  formed  is  dissolved  in  strong  alcohol  anil  decom- 
posed with  hydrogen  sulphide.  On  evaporation  the  free  acid  is 
obtained. 

Cholalic  Acid. — Cholalic  acid  is  the  principal  biliary  acid  which 
is  formed  in  the  liver,  and  to  its  presence  in  the  molecule  ot  glyoo- 
cholic  acid  and  taurocholic  acid  Pettenkofer's  reaction  is  due.  It  is 
a  product  of  the  specific  activity  of  the  liver-cells,  and  is  normally 
not  found  in  any  other  tissues  or  organs  of  the  animal  body.  In 
the  intestinal  contents,  however,  it  may  be  encountered,  as  such, 
even  in  health,  and  in  cases  of  jaundice  it  has  been  observed  also  in 
the  urine. 

A-  T  have  already  shown,  cholalic  acid  is  liberated  when  glyco- 
cholic  acid  or  taurocholic  acid  i-  decomposed  with  alkalies  or  acids, 
under  the  application  of  heat.  Its  crystalline  form  differs  accord- 
ing to  the  medium  in  which  crystallization  has  taken  place.  From 
it-  alcoholic  solution  it  separates  out  in  the  form  of  rhombic 
tetrahedra  or  octahedra,  which  contain  one  molecule  i>f  alcohol  as 
li,,uid  of  crystallization,  C^H^Oj  0aH6O.  On  prolonged  boiling 
with  water  the  crystal-alcohol  can  be  removed.  When  dissolved  in 
dilute  boiling  acetic  acid  cholalic  acid  takes  up  on.'  molecule  of 
water,  and  can  be  obtained  from  such  solution-  in  the  form  of 
rhombic  plates  or  prisms,  <  '..,1 1  ,,<  >,II.O.  On  exposure  to  the  ait- 
tin'  crystals  in  either  case  Boon  become  opaque.  They  melt  al  a 
temperature  of  195  < '.  The  free  acid  is  readily  soluble  in  alcohol, 
with  difficulty  bo  in  water,  and  is  almosl  insoluble  in  ether. 

According  to  Mylius,  it  is  a  monobasic  alcoholic  acid,  and  contains 
one  secondary  and  two  primary  alcohol  groups,  as  represented  by 

CH.OH 
the  formula:   C    II        MII..OH)..      It   combines  with  alkalies  and 

cool  I 
alkaline  earths,  a-  also  with  the  heavy  metals,  to  form  salts.     Its 
compounds  with  the  alkalies  are  readily  soluble  in  water,  but   with 


152  THE  DIGESTIVE  FLUIDS. 

some  difficulty  in  alcohol.  The  barium  salt  is  somewhat  soluble  in 
water,  and,  like  the  lead  salt,  soluble  in  hot  alcohol.  The  calcium 
salt  is  slightly  soluble  in  boiling  alcohol.  Concentrated  solutions  of 
the  alkaline  hydrates  and  carbonates  precipitate  the  alkaline  salts 
from  their  solutions  in  the  form  of  an  oily  material  which  becomes 
crystalline  on  cooling.  The  salts,  like  the  free  acid,  are  dextro- 
rotatory. 

On  prolonged  boiling  with  acids  or  at  a  temperature  of  200°  C. 
cholalic  acid  loses  two  molecules  of  water  and  is  transformed  into 
dyslysin,  as  shown  in  the  equation  : 

C24H40O5  =  C24H3603  +  2H20 

Cholalic  Dyslysin. 

acid. 

The  same  result  is  brought  about  through  the  influence  of  various 
bacteria,  and  there  can  be  no  doubt  that  the  dyslysin  which  is  con- 
stantly encountered  in  the  stools  is  referable  to  the  normal  processes 
of  putrefaction,  which  take  place  in  the  intestinal  canal. 

Choloidinic  acid,  C^H^Oj,  probably  represents  an  intermediary 
product  which  is  formed  during  this  process,  and  may  be  regarded 
as  a  primary  anhydride  of  cholalic  acid. 

The  common  dyslysin  which  is  met  with  in  the  feces  is  amorphous, 
and  is  insoluble  in  water  and  in  dilute  solutions  of  the  alkalies. 

On  oxidation  with  permanganate  cholalic  acid  first  yields  dehydro- 
cholalic acid,  and  then  bilianic  acid,  together  with  isobilianic  acid. 
These  changes  may  be  represented  by  the  equations  : 

(1)  C24H40O5     +    30    =    C24H3(05     +    3H20 
Cholalic  Dehydrocholalic 

acid.  acid. 

(2)  C24H3405     +     30    =    C24H3408 
Dehydrochol-  Bilianic 

alic  acid.  acid. 

On  further  oxidation  another  acid  has  been  obtained,  which  is 
termed  cilianic  acid,  and  which    is   said  to    have  the  composition 

Dehydrocholalic  acid  also  results  on  oxidizing  cholalic  acid  with 
nitric  acid ;  but  it  is  to  be  noted  that  on  further  oxidation  a  new 
acid  results,  which  is  known  as  choleocamphoric  acid,  and  is  thought 
to  be  isomeric  with  camphoric  acid.  Its  formula  is  given  as  CI0H16O4. 
On  oxidation  with  potassium  bichromate  and  sulphuric  acid,  on  the 
other  hand,  Tappeiner  claims  to  have  obtained  cholesteric  acid  (not 
to  be  confounded  with  cholesterinic  acid,  see  below),  C12H1607 ; 
'pyrocholesteric  acid,  C,,H1607;  cholanic  acid,  C20H28O6;  as  also 
palmitic,  stearic,  and  acetic  acids. 

On  reduction,  as  during  the  process  of  putrefaction,  cholalic  acid 
may  give  rise  to  the  formation  of  desoxy cholalic  acid,  C24H40O4.  On 
boiling  with  concentrated  solutions  of  the  alkaline  hydrates  a  mixt- 
ure of  the  corresponding  salts  of  formic  acid,  acetic  acid,  propionic 
acid,  and  palmitic  acid  is  obtained,  and  it  is  interesting  to  note  that 
the  latter,  like  cholalic  acid,  gives  Pettenkofer's  reaction. 


THE  BILE.  153 

Hyocholalic  acid  and  ehenocholalic  acid,  which  result  from  the 
decomposition  of  hyotaurocholic  acid,  hyoglycocholic  acid,  and 
chenotaurocholic  acid,  respectively,  in  their  general  properties  and 
reactions  are  closely  analogous  to  the  common  form  of  cholalic  acid 
that  has  just  been  considered.  Like  the  latter,  they  are  trans- 
formed in  the  intestinal  tract  into  the  corresponding  dyslysins, 
and  these  in  turn  yield  the  original  acids  on  heating  with  sodium 
hydrate  solution.  Both  are  soluble  in  alcohol  and  ether,  but 
insoluble  in  water. 

Isolation  of  Cholalic  Acid  from  the  Bile. — Platner's  bile,  obtained 
from  the  ox,  is  boiled  for  twenty-four  hours  with  barium  hydrate. 
The  cholalic  acid  which  is  thus  set  free  is  precipitated  by  adding 
an  excess  of  hydrochloric  acid.  It  is  then  washed  with  water, 
dissolved  in  a  dilute  solution  of  sodium  carbonate,  and  repre- 
cipitated  with  hydrochloric  acid.  The  precipitate  is  covered  with 
ether  and  allowed  to  stand,  when  crystallization  will  gradually  occur. 
The  crvstals  are  freed  from  liquid  as  far  as  possible  by  nitration  with 
the  suction-pump,  and  are  redissolved  in  hot  alcohol.  The  solution 
is  diluted  with  water  until  a  persisting  turbidity  develops,  and  is 
then  set  aside  in  a  cold  place  until  crystallization  is  complete. 

Choleic  Acid. — When  the  biliary  acids  of  ox-bile  are  decomposed, 
as  just  described,  a  small  amount  of  choleic  acid,  C24H4u04,  is  always 
found  associated  with  common  cholalic  acid.  On  oxidation  it  fii'st 
yields  drln/drocholeic  acid,  C24H3404,  and  then  eholanie  acid,  C24H34Or 
It  is  possibly  identical  with  desoxycholalic  acid  (see  above).  The 
substance  has  also  been  found  in  human  bile. 

Fellic  Acid. — This  acid  has  been  obtained  together  with  common 
cholalic  acid  and  choleic  acid  from  human  bile,  where  it  possibly 
exists  in  combination  with  glycocoll  and  taurin.  Its  amount  is 
always  small.  With  Pettenkofer's  test  it  gives  a  reddish-blue 
color.  On  heating,  it  is  decomposed,  with  the  formation  of  vapors 
which  are  stronglv  suggestive  of  turpentine.  Its  formula  is  given 
as  a{H,„04. 

Lithofellic  Acid. — Lithofellic  acid  is  a  substance  which  is  closely 
related  to  the  cholalic  acids  just  described,  and  is  found  in  certain 
gastro-intestinal  concretions  of  various  ruminants.  It  forms  the 
greater  portion  of  the  oriental  Bezaar  stones,  which  are  obtained 
from  the  stomach  of  the  wild  goat  and  antelope.  It  gives  Petten- 
kof'er'-   reaction,  and  is  said  to   have  the  formula  C2(JII.ir04. 

Taurin. — As  has  been  pointed  out,  taurin  is  not  exclusively 
found  in  the  bile,  but  occurs  also  in  the  lungs  and  kidneys  and  in 
the  muscle-tissue  of  many  animals,  and  notably  in  that  of  the  verte- 
brates. While  it  is  unquestionably  of  albuminous  origin,  and  prob- 
ably represents  an  intermediary  product  in  the  katalysis  of  the 
organic  sulphur    compounds  of  the    body,  it    has    thus    far    not    been 

obtained  from  tin-  source  by  artificial  means.     Unlike  the  loosely 

combined  sulphur  of  the  nlbiiminoiis    molecule,  the  sulphur  which  is 
present  in  the  taurin    molecule  cannot    be   split    oil'  on    boiling  with 


154  THE  DIGESTIVE  FLUIDS. 

dilute  alkalies.  Its  separation,  indeed,  necessitates  the  complete 
destruction  of  the  taurin,  viz.,  the  albuminous  material,  by  boiling 
with  a  concentrated  solution  of  sodium  hydrate  or  by  fusing  with 
potassium  hydrate  and  potassium  nitrate.  Potassium  sulphite,  acetic 
acid,  ammonia,  and  hydrogen  thus  result  in  the  first  case,  while  in  the 
latter  sulphates,  carbon  dioxide,  ammonia,  and  water  are  obtained. 
Taurin  can  be  formed  synthetically  by  heating  ammonium-ox  v- 
ethyl-sulphonate  to  a  temperature  of  230°  C,  or  from  ammonia  and 
chlor-ethyl-sulphonic  acid,  as  represented  by  the  equations : 

/C2H4C1  /C2H4.NH2 

NH3  +  S02<  =  SO/  +  HC1 

xOH  X)H 

Chlor-ethyl-sul-  Taurin. 

phonic  acid. 

/C2H4.OH  /C2H4.NH2 

S02<  =  S02<  +  H20 

xO.NH4  X)H 

Ammonium  oxy-  Taurin. 

ethyl-sulphonic 
acid. 

It  can  hence  be  regarded  as  amido-ethyl-sulphonic  acid  (viz.r 
amido-isethionic  acid).  It  crystallizes  in  the  form  of  four-  or  six- 
sided  prisms,  and  is  fairly  soluble  in  hot  water,  slightly  soluble  in 
common  alcohol,  and  insoluble  in  absolute  alcohol  and  ether.  It 
has  a  markedly  acid  character,  and  accordingly  does  not  combine 
with  acids,  but  with  alkalies,  to  form  salts.  Its  compound  with 
mercuric  oxide  is  quite  insoluble. 

Whether  or  not  a  relation  exists  between  taurin  and  cystin  is  as- 
yet  unknown.  The  latter  has  thus  far  only  been  observed  under 
conditions  which  must  be  regarded  as  abnormal,  and  it  would  be 
interesting  to  ascertain  whether  in  such  cases  the  formation  of  taurin 
is  possibly  diminished  or  even  suspended. 

Isolation  of  Taurin. — Taurin  is  most  conveniently  prepared  from 
the  bile  of  animals  in  Which  taurocholic  acid  is  present.  To  this 
end,  the  fluid  is  boiled  for  several  hours  with  hydrochloric  acid. 
The  dy  sly  sin  and  choloidinic  acid  are  filtered  off.  The  filtrate  is 
concentrated  on  a  water-bath  to  a  small  volume,  and  freed  from  the 
sodium  chloride  and  other  substances  that  have  separated  out,  by 
filtering  while  still  -warm.  The  liquid  is  then  evaporated  to  dryness 
and  the  residue  extracted  with  strong  alcohol,  which  dissolves  any 
glycocoll  hydrochlorate  that  may  be  present,  while  the  taurin  re- 
mains behind.  This  is  then  dissolved  with  a  little  warm  water,  fil- 
tered while  still  warm,  and  treated  with  an  excess  of  warm  alcohol. 
The  resulting  precipitate  is  immediately  filtered  off.  In  the  filtrate 
the  taurin  crystallizes  out  on  cooling,  and  can  be  identified  by  the  form 
of  its  crystals,  their  solubility  in  water  and  insolubility  in  alcohol,. 
and  the  formation  of  potassium  sulphate  when  fused  with  potassium 
hydrate  and  potassium  nitrate. 

Should  glycocholic  acid  be  present  in  the  bile  at  the  same  time, 
this  is  likewise  decomposed  on  boiling  with  hydrochloric  acid.     The 


THE  BILE.  155 

hydrochlorate  of  glycocoll  which  thus  results  is  found  in  the  first 
alcoholic  extract.  To  isolate  the  glvcocoll  as  such,  the  solution  is 
evaporated  to  dryness,  the  residue  dissolved  in  water  and  treated 
with  plumbic  hydrate.  On  filtering,  the  solution,  which  contains 
the  lead  salt  of  glvcocoll,  is  decomposed  with  hydrogen  sulphide. 
The  resulting  lead  sulphide  is  filtered  off',  and  the  filtrate  concen- 
trated until  crystallization  occurs.  The  crystals  are  then  dissolved 
in  water  and  decolorized  with  animal  charcoal,  and  the  solution  is 
evaporated  until  the  crystals  again  separate  out. 

Glycocoll. — Glvcocoll  is  now  known  to  be  a  constant  decomposi- 
tion-product of  most  albumins,  but  is  formed  in  especially  large 
amounts  during  the  hydrolytic  decomposition  of  collagen  and 
spongin.  From  this  fact  and  its  sweetish  taste  it  is  also  known 
as  glucin  or  collagen-sugar  (Leimzucker).  It  is  one  of  the  most 
important  decomposition-products  which  are  formed  in  the  nitro- 
genous metabolism  of  the  animal  body,  and  in  part  at  least  gives 
rise  to  the  formation  of  urea  in  mammals,  and  to  uric  acid  in  birds 
and  reptiles.  Another  portion,  as  we  have  seen,  is  eliminated  in 
the  bile  in  combination  with  cholalic  acid;  while  a  third  portion 
appears  in  the  urine  in  combination  with  benzoic  acid  and  phenyl- 
acetic  acid,  as  hippuric  acid  and  phenaceturic  acid,  respectively  (which 
see). 

As  we  have  seen,  glvcocoll  is  amido-acetic  acid.  The  pure  sub- 
stance crystallizes  in  the  form  of  colorless  rhombohedra  or  of  four- 
sided  prisms,  which  are  readily  soluble  in  water,  with  difficulty  so 
in  warm  alcohol,  and  insoluble  in  absolute  alcohol  and  ether.  It 
combines  with  acids  and  alkalies  to  form  salts.  The  most  important 
of  these  are  the  hydrochlorate,  which  is  soluble  in  water  and  alcohol, 
and  the  copper  salt,  which  results  when  a  boiling  solution  of  glycocoll 
is  added  t<>  freshly  precipitated  cupric  hydrate;  the  hydrate  is  thus 
dissolved,  and  after  concentrating  the  solution  blue  needles  of  the 
copper  salt  separate  out  on   cooling. 

Isolation. — Glvcocoll  can  be  obtained  from  the  bile  of  those 
animals  in  which  glycocholic  acid  is  found,  as  described  above;  or 
it  may  be  prepared  from  hippuric  acid  by  decomposing  this  by  boil- 
ing with  dilute  anlphuric  acid.  On  cooling,  the  benzoic  acid  that 
has  separated  out  i-  filtered  oil',  the  filtrate  is  concentrated  and  ex- 
tracted with  ether  to  remove  such  benzoic  acid  as  still  remains  in 
solution  ;  the  sulphuric  acid  is  removed  with  barium  carbonate,  and 
the  filtrate  i-  evaporated  until  crystals  of  glycocoll  begin  to  separate 
out  (see  al-o  pages  192  and  259). 


The   Bile-pigments. 

The  bile-pigments   which    have  thus  Car  been  obtained  from  the 
bile    it-elf  or  from    biliary  concretion-  are  bilirubin,  biliverdin,  bili- 

prasin,  bilifuscin,  and  other-  which  are  less  well  known. 


156  THE  DIGESTIVE  FLUIDS. 

Perfectly  fresh  hepatic  bile,  in  contradistinction  to  that  which  is 
found  in  the  gall-bladder,  contains  only  one  pigment,  bilirubin,  from 
which  all  other  forms  are  derived.  Such  bile  is  of  a  golden-yellow 
color,  while  bladder-bile  usually  presents  an  olive-brown  color,  owing 
to  the  simultaneous  presence  of  its  nearest  oxidation-product,  bili- 
verdin.  A  grass-green  color  is  observed  when  the  latter  predomi- 
nates or  is  exclusively  present. 

Bilirubin. — Bilirubin  is  now  known  to  result  from  the  decompo- 
sition of  haematin,  and  normally  constitutes  a  specific  product  of  the 
activity  of  the  hepatic  cells.  It  appears,  however,  that  the  power 
of  transforming  the  blood-pigment  into  bilirubin  is  common  to  other 
tissues  as  well,  if  we  regard  the  haematoidin  of  Virchow,  which  is 
so  often  found  in  old  extravasations  of  blood,  as  identical  with  bili- 
rubin. That  this  is  actually  the  case  seems  now  undoubted.  Under 
normal  conditions,  however,  the  liver  is  apparently  the  only  organ 
of  the  body  in  which  the  formation  of  bilirubin  takes  place. 
"Whether  or  not  the  final  dissolution  of  disintegrating  red  corpuscles 
also  occurs  at  this  place  has  not  been  decided,  but  the  liver  is 
manifestly  capable  of  retaining  the  haemoglobin  which  is  thus  set 
free.  If  a  moderate  amount  of  a  solution  of  haemoglobin  is  injected 
into  the  bloodvessels  of  an  animal,  an  increased  elimination  of 
bilirubin  results,  while  the  blood-pigment  does  not  appear  in  the 
urine.  If  larger  amounts  are  injected,  the  resulting  bilirubin  ap- 
parently cannot  be  removed  with  sufficient  rapidity  through  the 
biliary  ducts.  Resorption  of  the  pigment  then  takes  place  through 
the  lymph-vessels,  jaundice  results,  and  the  bile-pigment  appears  in 
the  urine.  If  excessive  amounts  of  haemoglobin  finally  are  injected, 
the  liver  is  manifestly  incapable  of  retaining  all  the  pigment  which 
reaches  the  organ,  jaundice  results,  and  both  the  bile-pigment  and 
the  blood-pigment  appear  in  the  urine.  Even  in  such  extreme 
cases  the  remaining  tissues  of  the  body  do  not  participate  in  the 
formation  of  bilirubin.  This  has  been  conclusively  demonstrated 
by  Minkowski  and  Xaunyn.  These  observers  have  shown  that  while 
in  normal  geese  poisoning  with  hydrogen  sulphide,  which  causes 
an  extensive  dissolution  of  the  red  corpuscles,  invariably  leads  to 
jaundice  and  the  appearance  of  bile-pigment  in  the  urine,  the 
previous  removal  of  the  liver  prevents  such  an  occurrence,  and 
results  in  simple  hemoglobinuria.  In  mammals  such  crucial  tests 
unfortunately  cannot  be  applied,  but  there  is  no  reason  to  suppose 
that  different  conditions  there  exist.  The  possible  occurrence  of  a 
haematogenic  icterus,  in  contradistinction  to  a  hepatogenic  icterus,  is 
thus  rendered  extremely  improbable,  and  it  is  scarcely  warrantable 
to  point  to  the  development  of  bilirubin  from  blood-pigment  in  the 
tissues  of  the  organs,  as  indicating  the  possibility  of  such  a  trans- 
formation in  the  circulating  blood. 

Of  the  manner  in  which  this  transformation  is  effected  m  the  liver 
we  know  nothing.  "We  may  imagine,  however,  that  the  oxyhemo- 
globin is  here  first  decomposed  into  its  albuminous  component — 


THE  BILE.  157 

globin — and  into  hsematin,  which  latter  then  passes  over  into  bili- 
rubin, as  shown  in  the  equation  : 

LVJI3.N  AFe  +  2H20  —  Fe  =  C32H36N4<  >6 
Haematin.  Bilirubin. 

This  reaction,  it  will  be  noted,  is  also  supposed  to  express  the 
formation  of  haematoporphyrin  from  hsematin  (see  page  837).  The 
two  substances,  indeed,  are  now  quite  generally  regarded  as  isomeric. 
As  regards  the  size  of  the  molecule,  however,  opinions  differ,  and  it 
is  possible,  as  Nencki  and  Sieber  suppose,  that  the  molecule  of  bili- 
rubin, as  well  as  of  haematoporphyrin,  is  only  half  as  large  as 
expressed  above.  In  that  case,  of  course,  the  equation  would  have 
to  be  written  : 

C32H32X  AFe  +  2H20  —  Fe  =  2C16H18N2Os 

On  reduction  with  nascent  hydrogen  bilirubin  is  transformed  into 
hydrobilirubin,  as  is  shown  by  the  equation  : 

C32H36X405(viz.,  2C16H18N203)  +  H20  +  2H  =  C32H40N4O7 

Uydrobilirubin. 

This  actually  takes  place  in  the  intestinal  canal,  where  nascent 
hydrogen  is  constantly  being  formed  through  the  action  of  various 
bacteria,  as  during  the  process  of  lactic  acid  fermentation.  During 
intra-uterine  life,  however,  where  no  bacteria  are  found  in  the 
intestinal  tract,  bilirubin  appears  in  the  feces  as  such  (meconium). 

According  to  some  observers,  hydrobilirubin  is  thought  to  be 
identical  with  the  febrile  urobilin  of  Jaffe,  which  is  met  with  in  the 
urine  in  various  febrile  diseases,  and  exceptionally  also  under  normal 
conditions,  but  it  is  said  to  differ  from  the  normal  urobilin  which 
represents  the  principal  coloring-matter  of  the  urine  in  health. 
This  question,  however,  still  awaits  solution.  The  possible  identity 
also  of  hydrobilirubin  and  stercobilin,  which  is  the  principal  pig- 
ment that  is  found  in  the  feces,  has  not  been  definitely  established 
(see  also  pages  207  and  291). 

In  the  crystalline  state  bilirubin  occurs  in  the  form  of  reddish- 
yellow  rhombic  platelets  with  rounded  angles,  which  arc  soluble  in 
benzol,  carbon  disulphide,  amyl  alcohol,  glycerin,  the  fatty  oils,  and 
especially  in  warm  chloroform.  In  alcohol  and  ether  they  are  but 
little  soluble,  and  in  water,  as  also  in  Platner's  bile,  they  are  insol- 
uble On  spectroscopic  examination  its  solutions  do  not  give  rise  to 
any  specific  bands  of  absorption,  but  to  a  continuous  absorption 
which  extend-  from  the  red  to  the  violet  end. 

Bilirubin  as  such  is  a  weak  acid,  bilirubinic  arid,  and  combines 
with  bases  to  form  salts,  which  for  the  raosl  part  are  either  insoluble 
or  only  slightly  Boluble  in  water  and  insoluble  in  chloroform.     Its 

salts  with  the  alkalies,  however,  arc  soluble  in  solution-  of  the  alka- 
line hydrates  and  carbonates.  In  the  bile  bilirubin  i-  largely  present 
as  neutral  bilirubinate  of  sodium,  and  is  held  in  solution  owing  to 
the  presence  of  alkaline  carbonates. 


158  THE  DIGESTIVE  FLUIDS. 

When  perfectly  fresh  bile  of  a  golden-yellow  or  olive-brown  color 
is  exposed  to  the  air  for  a  while,  it  will  be  noted  that  the  fluid  grad- 
ually assumes  a  bright-green  color,  owing  to  a  transformation  of  the 
bilirubinate  into  bidverdinate  of  sodium.  Free  bilirubin,  according 
to  Dastre  and  Floresco,  does  not  absorb  oxygen  and  is  thereby 
transformed  into  biliverdin,  as  has  been  supposed.  The  same 
observers  state  that  on  careful  oxidation  bilirubin  can  be  trans- 
formed into  biliprasin,  which  presents  a  green  color  as  such,  while 
its  sodium  salt,  sodium  biliprasinate,  is  yellowish  brown.  On 
further  oxidation  the  biliprasin  then  yields  biliverdin,  viz.,  its  cor- 
responding salt. 

According  to  former  views,  the  relation  existing  between  bilirubin 
and  biliprasin  were  represented  by  the  equations  given  below ;  but 
it  is,  of  course,  manifest  that  these  are  no  longer  tenable  if  the  work 
of  Dastre  and  Floresco  should  be  confirmed. 

C16H18N203  +  0  =  C18H18N204 

Bilirubin.  Biliverdin. 

C16H18N,03  +  H20  =  C16H20NA 

Bilirubin.  Bilifuscin. 

ClfiH20N2O4  +  H20  +  O  =  ClfiH22N206 
Bilifuscin.  Biliprasin. 

The  formula  of  biliprasin,  as  here  indicated,  would,  moreover,  be 
an  impossibility.  As  a  matter  of  fact,  these  formulae  cannot  be 
regarded  as  definitely  established  ;  and  according  to  some  observers, 
the  molecule  of  biliverdin  is  only  one-half  as  large  as  represented 
above.  Much  work  still  remains  to  be  done  in  this  connection,  but 
we  know  at  least  that  biliverdin  constitutes  a  normal  oxidation- 
product  of  bilirubin.  From  biliverdin  Kiister  claims  to  have 
obtained  a  new  oxidation-product,  which  he  termed  biliverdinic  acid, 
but  which  must  not  be  confounded  with  the  substance  of  the  same 
name  referred  to  above.  This  body  has  the  formula  C8H9N04,  and 
is  apparently  identical  with  the  dibasic  hsematinic  acid,  which  results 
from  haematin  directly,  and,  like  this,  yields  a  substance  of  ihe  com- 
position C8H805,  viz.,  the  anhydride  of  the  dibasic  hsematinic  acid 
C8H10O6.  In  this  manner  the  origin  of  bilirubin  from  the  coloring- 
matter  of  the  blood  is  still  further  shown. 

If  bile  containing  bilirubin  is  filtered  through  Swedish  filter- 
paper,  and  a  drop  of  concentrated  nitric  acid  containing  a  trace 
of  nitrous  acid, is  placed  upon  the  paper,  which  is  colored  a  bright 
yellow,  a  play  of  colors  will  be  observed,  in  which  the  yellow  first 
turns  to  green,  then  to  blue,  to  violet,  to  red,  and  ultimately  to 
orange.  This  reaction  is  commonly  referred  to  the  formation  of 
various  oxidation-products  of  bilirubin,  and  is  very  characteristic. 
The  individual  products,  however,  which  thus  result  are  mostly  but 
imperfectly  known.  The  green  color,  of  course,  is  referable  to  the 
biliverdin.  The  blue  color  is  ascribed  to  bilicyanin  or  cholecyanin, 
and  the  final  orange  to  choletelin. 

Tests  for  Bilirubin. — Gmelin's  Test. — The  fluid  to  be  examined 


THE  BILE. 

is  treated  with  an  amount  of  concentrated  nitric  acid,  containing  a 
trace  or  nitrous  acid,  sufficient  to  form  a  layer  beneath  the  liquid  to 
be  tested,  when  in  the  presence  of  bilirubin  the  color-play  referred  to 
will  be  observed  at  the  zone  of  contact ;  the  green  will  be  noticed 
nearest  the  bile-cuntaining  solution,  and  the  orange  in  the  upper 
portion  of  the  nitric  acid.  Various  modifications  of  this  reaction 
have  been  proposed,  such  as  the  one  described  above. 

The  test  is  exceedingly  sensitive,  and  is  said  to  indicate  the   | 
ence  of  bilirubin  in  a  dilution  of  1      v  \     The  green  color  which 

develops  is  the  most  characteristic,  but  a  reddish  violet  must  also 
occur. 

Huppeet'-  Test. — A  few  cubic  centimeters  of  the  solution  to 
lie  examined  are  precipitated  with  barium  chloride  and  ammonia. 
The  precipitate  is  washed  with  water  and  suspended  in  a  small 
amount  of  alcohol  that  has  been  acidulated  with  sulphuric  acid. 
This  mixture  is  then  boiled  for  a  few  minutes,  when  in  the  presence 
of  bilirubin  a  bright  emerald-ofreen   color  develop- . 

Smith's  Te-t. — A  -mall  amount  of  the  fluid  is  placed  in  a  test- 
tube  and  treated  with  a  few  cubic  centimeters  of  tincture  of  iodine 
which  has  been  diluted  with  alcohol  in  the  proportion  of  1  :  10,  so 
as  to  form  a  layer  above  the  fluid  to  be  examined.  In  the  presence 
of  bilirubin  a  distinct  emerald-green  ring  will  develop  at  the  zone 
of  contact. 

According  to  some  observers,  the  green  color  which  thus  results 
when  bilirubin  is  treated  with  inline  is  not  referable  to  the  forma- 
tion of  biliverdin,  but  to  a  substitution-  or  addition-product  of  bili- 
verdin  with  iodine.  This,  however,  is  denied  by  others,  and  Jolles 
has  recently  shown  that  the  iodine  merely  acts  as  an  oxidizinsr  agent, 
and  that  true  biliverdin  is  thus  formed,  as  indicated  by  the  equation  : 

C^H^A  -  -21  -  H,0  =  C16H„XA  -  2HI. 

Spectroscopic-  Tebt. — If  a  dilute  solution  of  sodium  bilirubi- 
nate in  water  is  treated  with  an  exces-  of  ammonia  and  a  small 
amount  of  a  solution  of  chloride  of  zinc,  the  liquid  at  first  turns 
a  deep  orange,  but  subsequently  becomes  olive  brown,  and  finally 
green.  If  this  solution  i-  examined  spectroscopically,  it  will  be 
•1  that  the  violet  and  blue  portions  of  the  spectrum  are  at  first 
quite  dark,  but  subsequently  the  bands  presented  by  an  alkaline 
-olution  of  bilicyanin  become  apparent,  and  notably  the  one  between 
I  md  D,  near  C  see  below).  The  test  i-  said  to  be  very  g 
1 1  immarsten). 

Isolation  of  Bilirubin. — Bilirubin  i-  most  conveniently  obtained 
from  the  biliary  concretions  which  an-  -<>  often  found  in  the  srall- 
Madder  -.f  cattle,  and  which  consist  almost  entirely  of  the  calcium 
-alt  of  the  pigment.  They  ;ire  finely  powdered  and  extracted  with 
ether  and  then  with  hot  water,  bo  a-  to  remove  the  cholesterin  and 
the  biliary  acids  which  an-  present.  The  remaining  material  i- 
treated  with  hydrochloric  acid,  soaa  to  liberate  die  pigment.     It  i- 


160  THE  DIGESTIVE  FLUIDS. 

then  washed  free  from  acid  with  water,  and  subsequently  with  abso- 
lute alcohol,  to  remove  the  water  and  any  biliverdin  that  may  be 
present.  The  pigment  remains,  and  is  now  dissolved  with  boiling 
chloroform.  From  this  solution  the  chloroform  is  distilled  off,  the 
resklue  extracted  with  absolute  alcohol,  so  as  to  remove  any  bili- 
fuscin,  and  the  remaining  bilirubin  dissolved  in  a  small  amount  of 
chloroform  and  precipitated  with  alcohol.  This  procedure  is 
repeated  until  the  substance  has  been  obtained  in  pure  form  ;  it 
is  then  allowed  to  crystallize  out  from  its  chloroform  solution  on 
cooling. 

Biliverdin. — Biliverdin  is  found  in  the  bladder-bile  of  many  ani- 
mals together  with  bilirubin,  and  is  especially  abundant  in  certain 
herbivora,  where  the  bile  frequently  presents  a  bright  grass-green 
color.  It  is  said  to  occur  also  in  the  placenta  of  the  bitch,  in  the 
shells  of  certain  mollusks,  and  in  birds'  eggs.  Its  relation  to  bili- 
rubin has  already  been  considered.  In  the  bile  it  is  present  princi- 
pally in  the  form  of  its  sodium  salt,  and,  like  bilirubin,  the  free 
pigment  possesses  acid  properties ;  this  is  termed  biliverdinic  acid, 
but  should  not  be  confounded  with  the  acid  of  the  same  name 
which  Kuster  obtained  from  biliverdin  on  oxidation  with  sodium 
chromate.  In  acid  bile  biliverdin  is  found  as  such.  Unlike 
bilirubin,  the  free  pigment  is  readily  soluble  in  normal  as  well  as  in 
neutral  and  acid  bile.  It  is  insoluble  in  water,  ether,  and  chloro- 
form, but  dissolves  in  alcohol,  glacial  acetic  acid,  and  solutions  of 
the  alkalies.  From  the  latter  it  is  precipitated  by  the  salts  of  the 
alkaline  earths  and  the  heavy  metals,  as  also  by  acids.  On  treating 
an  alcoholic  solution  of  biliverdin  with  ammoniacal  chloride  of  zinc 
solution  the  fluid  exhibits  a  green  fluorescence.  The  green  color  of 
the  pigment  is  changed  to  yellow  if  its  solution  in  acid  alcohol  is 
treated  with  zinc.  If  chlorine-water  is  added  instead,  a  blue  color 
develops  at  the  bottom  of  the  liquid,  and  above  it  layers  present- 
ing a  violet,  a  red  and  a  yellow  color  will  be  observed.  On  adding 
an  excess  of  chlorine-water  the  solution  is  decolorized. 

Pure  biliverdin,  like  bilirubin,  gives  no  characteristic  band  of 
absorption  in  alkaline  solution.  In  acid  solution,  however,  or  in 
pure  alcoholic  solution,  an  indistinct  band  is  observed  at  D,  and  one 
that  is  more  pronounced  near  F. 

The  substance  is  amorphous,  or  at  least  cannot  be  obtained  in  a 
pronounced  crystalline  form. 

On  reduction,  biliverdin  is  supposedly  transformed  into  bilirubin, 
though  this  is  denied  by  some  observers. 

On  oxidation  with  nitric  acid  biliverdin  gives  rise  to  the  various 
colors  which  have  already  been  described,  beginning  with  blue.  It 
gives  Huppert's  reaction  directly. 

Isolation. — To  prepare  biliverdin,  it  is  most  convenient  to  start 
with  a  solution  of  bilirubin  in  the  form  of  its  sodium  salt  which  has 
been  exposed  to  the  air  until  the  original  golden-yellow  color  has 
changed  to  a  brownish  green.     The  biliverdin  is  then  precipitated 


THE  BILE.  161 

by  adding  an  excess  of  hydrochloric  acid.  It  is  filtered  off,  washed 
free  from  all  acid,  dissolved  in  absolute  alcohol,  and  precipitated  by 
copiously  diluting  with  water.  Any  bilirubin  that  may  be  present 
is  removed  by  extracting  with  chloroform. 

Biliprasin.— Biliprasin  as  such,  and  biliprasinate  of  sodium, 'ac- 
cording to  Dastre  and  Floresco,  are  also  found  in  normal  bladder- 
bile,  and,  as  has  been  indicated,  represent  intermediary  products  of 
oxidation  between  bilirubin  and  biliverdin. 

The  sodium  salt,  according  to  these  observers,  is  a  yellowish-brown 
pigment,  and  can  be  transformed  into  biliprasin  through  the  agency 
of  mineral  acids,  of  acetic  acid,  and  even  of  carbonic  acid.  On  ex- 
posure to  the  air  it  passes  over  into  the  corresponding  biliverdinate. 
To  its  presence  the  yellow  color  of  the  bile  of  calves  and  of  other 
animals  is  supposedly  due. 

Biliprasin  itself  is  green,  but  can  be  transformed  into  the  yellow 
salt  by  adding  a  few  drops  of  an  alkali,  and  this  in  turn  vields  the 
green  pigment  on  treating  with  an  acid.  This  reaction,  according 
to  Dastre  and  Floresco,  explains  the  fact  that  yellow  bile  can  become 
green  without  oxidation,  viz.,  without  the  formation  of  biliverdin. 
According  to  older  ideas,  however,  biliprasin  is  an  oxidation-product 
of  biliverdin,  and  is  supjjosed  to  result  from  this  with  the  inter- 
mediary formation  of  bilifuscin,  as  has  alreadv  been  outlined. 

Bilifuscin  is  said  to  occur  in  gall-stones  together  with  bilirubin. 
The  formula  which  has  been  ascribed  to  it  is  that  of  bilirubin  plus 
one  or  two  molecules  of  water,  viz.,  C82HwN"408  or  C1HH20N,O4.  It 
is  of  a  dark-brown  color,  and  is  soluble  in  alcohol  and  the  solutions 
of  the  alkaline  hydrates,  but  insoluble  in  water  and  chloroform. 
In  pure  form  it  does  not  give  Gmelin's  reaction. 

Bilicyanin,  or  cholecyanin,  is  the  blue  pigment  -which  is  formed 
during  the  oxidation  of  bilirubin  arid  biliverdin  with  nitric  acid.  It 
has  been   found   together  with   the  common   bile-pigments  in  gall- 

81 «  taken  from  man.     Its  neutral  and  alkaline  solutions  give  rise 

to  three  bands  of  absorption.  One  of  these  is  located  between  C 
and  I),  near  C;  another  about  D;  and  a  third  very  faint  band 
midway  between  Hand  E.  In  acid  solutions  two  bands  are  seen 
between  C  and  E.  On  treating  the  alcoholic  solution  of  the  pig- 
menl  with  an  ammoniacal  solution  of  zinc  chloride  a  distinct  fluor- 
escence is  obtained.  Its  formula  has  not  as  yet  been  determined, 
and,  according  to  some  observers,  indeed,  bilicyanin  does  nut  repre- 
sent a  separate  substance. 

Bilipurpurin.— This  term  has  been  applied  to  the  red  pigment 
which  is  formed  from  bilirubin  and  biliverdin  on  treating  with 
nitric  acid.  Nothing  is  known  of  its  properties  or  chemical  com- 
position. 

Choletelin,  or  bilixanthin,  is  generally  regarded  as  the  final 
oxidation-product  of  the  common  bile-pigments.  It  is  an  amorphous 
brown  substance,  which  i-  soluble  in  alcohol,  ether,  chloroform,  and 
1,1  solutions  of  the  alkaline  hydrates,  from  which  latter  it  can  be 

n 


162  THE  DIGESTIVE  FLUIDS. 

precipitated   by  the    addition    of    acids.     Its    formula  is    given  as 
C16H1?NA; 

Bilihumin  is  a  pigment  of  unknown  composition  that  has  been 
found  in  gall-stones.     It  is  insoluble  in  all  organic  solvents. 

Cholesterin. 

Cholesterin  is  not  exclusively  a  product  of  the  activity  of  the 
hepatic  cells,  but  is  found  in  other  tissues  as  well.  It  has  thus  been 
encountered  in  the  red  corpuscles  of  the  blood,  in  the  plasma,  in  the 
yolk  of  eggs,  in  the  semen,  and  in  the  secretion  of  the  sebaceous 
glands,  and  is  especially  abundant  in  nerve-tissue.  In  the  vegetable 
world  also  cholesterin  is  distributed.  The  liver  is  probably  the 
organ  through  which  the  substance,  wherever  formed,  is  eliminated. 
Ultimately  it  appears  in  the  feces.  In  the  urine  it  is  found  only 
under  exceptional  conditions,  and  then  only  in  very  small  amounts. 
Of  its  mode  of  formation  nothing  is  known,  but  it  is  interesting  to 
note  that  wherever  cholesterin  is  found  lecithin  is  likewise  observed. 
In  the  brain  a  considerable  amount  of  the  substance  occurs  in  com- 
bination with  a  fatty  acid,  cholic  acid,  from  which  it  can  only  be 
separated  by  saponification. 

The  amount  of  cholesterin  which  is  found  in  the  bile  represents 
about  2  per  cent,  of  the  total  solids.  Normally  it  is  held  in  solution 
owing  to  the  presence  of  the  biliary  acids,  but  under  pathologic  con- 
ditions it  may  separate  out  in  crystalline  form,  either  in  the  gall- 
bladder itself  or  in  the  larger  hepatic  ducts,  and  then  gives  rise  to 
the  formation  of  stones.  Of  the  origin  of  these  concretions  we  know 
little.  Very  often  they  contain  a  nucleus  of  epithelial  cells  or  of 
bacteria,  around  which  the  cholesterin,  together  with  a  variable 
amount  of  bile-pigment  and  mineral  salts,  becomes  deposited.  It  is 
possible  that  they  result  owing  to  a  temporary  absence  of  the  biliary 
acids,  but  this  is  only  a  supposition.  The  stones  which  are  usually 
found  in  man  are  for  the  most  part  very  rich  in  cholesterin,  while 
the  pigment-stones  which  are  so  common  in  cattle  are  less  frequently 
seen  in  the  human  being. 

The  common  cholesterin  which  is  found  in  the  animal  body  has 
the  composition  C27H45.OH  (Obermiiller),  and  is  usually  regarded  as 
a  monatomic  alcohol.  Of  its  structure,  however,  nothing  is  known. 
It  combines  with  fatty  acids  to  form  compound  ethers  which  are 
analogous  to  the  fats,  and  in  this  form  also,  as  has  been  shown 
(page"  67)  cholesterin  occurs  widely  distributed  in  the  animal 
world.  The  common  lanolin  of  wool-fat  thus  contains  large 
amounts  of  such  compound  ethers,  both  of  cholesterin  and  its 
isomeric  compound  isocholesterin.  On  treating  cholesterin  with 
concentrated  sulphuric  acid  the  substance  gives  rise  to  the  formation 
of  certain  hydrocarbons,  which  are  termed  cholesterilins,  and  which 
are  supposed  to  stand  in  a  close  relation  to  the  terpene  group.  With 
iodine  these  bodies  give  a  blue  color. 


THE  BILE.  163 

Cholesterin  usually  occurs  in  the  form  of  colorless,  transparent 
plates,  with  ragged  margins  and  angles,  which  are  very  characteristic. 
It  is  practically  insoluble  in  water,  dilute  acids  and  alkalies,  but  dis- 
solves with  ease  in  ether,  chloroform,  benzol,  and  in  boiling  alcohol. 
From  its  ethereal  solutions  it  crystallizes  out  in  the  form  of  fine 
needles.  It  is  further  soluble  in  the  essential  and  fatty  oils,  as  also 
in  the  presence  of  biliary  acids.     Its  crystals  melt  at  145°  C. 

Tests. — Salkowski's  Test. — A  few  crystals  of  cholesterin  are 
dissolved  in  a  small  amount  of  chloroform  and  treated  with  an 
equal  volume  of  concentrated  sulphuric  acid.  The  solution  of 
cholesterin  then  first  assumes  a  blood-red  color,  and  then  gradually 
turns  to  a  violet  red,  while  the  sulphuric  acid  appears  dark  red  and 
shows  a  green  fluorescence.  On  pouring  the  chloroform  into  a 
shallow  porcelain  dish  it  turns  violet,  then  green,  and  finally  becomes 
yellow. 

The  Test  of  Liebermann-Burckhard. — If  a  small  amount 
of  cholesterin  is  dissolved  in  about  2  c.c.  of  chloroform  and  is 
treated  with  10  drops  of  acetic  acid  anhydride,  and  subsequently 
with  concentrated  sulphuric  acid,  the  solution  at  first  assumes  a  red, 
then  a  blue,  and  finally  a  green  color.  The  latter  develops  at 
once  if  cholesterin  is  present  only  in  traces. 

Isolation. — To  prepare  cholesterin  for  purposes  of  study,  it  is 
most  convenient  to  isolate  the  substance  from  cholesterin  stones. 
To  this  end,  the  concretions  are  finely  pulverized  and  extracted 
with  boiling  water  and  then  with  boiling  alcohol.  From  the 
alcoholic  extract  the  cholesterin  crystallizes  out  on  cooling,  and  is 
then  boiled  with  an  alcoholic  solution  of  sodium  hydroxid,  so  as  to 
saponify  the  fats  which  are  at  the  same  time  present.  The  alcohol 
is  then  distilled  off  and  the  residue  extracted  with  ether,  which 
removes  the  cholesterin  and  leaves  the  soaps  behind.  On  evaporat- 
ing this  extract,  after  filtration,  the  substance  is  obtained  in  crystal- 
line form,  and  can  be  further  purified  by  recrystallization  from  a 
mixture  of  alcohol  and  ether. 

Other  Organic  Constituents  of  the  Bile. — In  addition  to  the 
bodies  already  described,  the  bile  contains  also  small  amounts  of 
lecithin,  of  palmitin,  stearin,  olein,  and  the  soaps  of  the  correspond- 
ing fatty  acids.  In  ox-bile  Lassar-Cohn  found  also  traces  of 
myristinic  acid,  CuH28021,  which  has  heretofore  only  been  observed 
in  the  spermaceti  of  whales.  We  further  find  traces  of  urea, 
and  occasionally  a  diastatic  ferment,  which  is  by  some  observers 
regarded  as  identical  with  ptyalin.  Its  presence,  however,  is  by 
no  means  constant,  and  it  can  scarcely  be  regarded  as  playing  a 
rdle  in  the  process  of  intestinal  digestion.  Larger  amounts  of  urea, 
according   to    Hammai-ten,  are   found   in   the  bile  of  the  shark  and 

the  sturgeon. 

In  decomposing  bile  cholin,  glycerin-phosphoric  acid  and  tri- 
incthvlaniin  mav  be  observed,  and  are  referable  to  the  decomposition 
of  lecithin  (see  pages  65  and  66). 


164  THE  DIGESTIVE  FLUIDS. 

The  Biliary  Iron. 

If  we  recall  the  origin  of  bilirubin  from  hsematin,  we  should 
expect  to  find  in  the  bile  the  iron  which  is  liberated  during  the 
decomposition  of  the  latter.  Traces  of  iron,  indeed,  are  constantly 
present,  principally  in  combination  with  phosphoric  acid.  The 
amount,  however,  which  is  thus  eliminated  is  far  too  small  to  repre- 
sent that  which  must  of  necessity  be  set  free.  Kunkel  thus  found 
that  while  100  parts  of  hsematin  correspond  to  9  parts  of  iron,  only 
1.4—1.5  parts  of  iron  appear  in  the  urine  for  every  100  parts  of 
bilirubin.  But  even  if  we  add  to  this  the  amount  which  is  eliminated 
in  the  urine  and  that  which  is  excreted  through  the  intestinal  mucosa, 
we  still  find  a  very  large  deficit,  and  we  are  accordingly  forced  to 
the  conclusion  that  the  greater  portion  of  the  iron  must  be  retained 
in  the  liver.  But  while  such  a  retention  must  of  necessity  occur,  we 
are  profoundly  ignorant  of  the  manner  in  which  it  is  accomplished. 
Of  the  form,  also,  in  which  the  iron  exists  in  the  liver  we  know  but 
little.  That  it  is  subsequently  utilized  in  the  construction  of  haemo- 
globin is  quite  likely,  but  not  proved.  Naunyn  and  Minkowski  have 
observed  that  following  poisoning  with  arseniuretted  hydrogen,  iron- 
containing  pigments  can  be  demonstrated  in  the  liver.  Latschen- 
berger  speaks  of  the  formation  of  choleglobins  as  antecedents  of  bili- 
rubin, and  of  the  simultaneous  appearance  of  iron-containing  melanins 
in  the  liver;  and  Neumann  has  demonstrated  the  presence  of  an 
organic  iron  pigment,  hsemosiderin,  in  old  extravasations  of  blood 
and  in  thrombi  together  with  hsematoidin ;  but  of  the  true  nature 
of  all  these  substances  we  practically  know  nothing.  It  is  possible 
that  the  globin,  which  must  of  necessity  be  liberated  during  the 
decomposition  of  hsematin,  takes  up  the  iron  which  is  set  free  from 
the  latter ;  but  this  also  is  a  supposition,  and  further  researches  in 
this  direction  are  urgently  needed. 


CHAPTER   VIII. 

THE  PROCESSES  OF   DIGESTION  AND  RESORPTION. 

In  the  preceding  chapter  we  have  considered  the  various  digestive 
fluids  which  are  concerned  in  the  transformation  of  those  food- 
stuffs that  are  incapable  of  resorption  as  such  into  material  which 
the  body  can  utilize  for  purposes  of  nutrition,  and  we  have  seen  that 
the  most  important  agents  which  are  here  concerned  belong  to  the 
class  of  the  non-organized  ferments.  In  the  present  chapter  we 
shall  study  the  action  of  these  various  substances  upon  the  different 
classes  of  food-stuffs  collectively  and  in  somewhat  greater  detail, 
ami  shall  incidentally  also  consider  the  resorption  of  the  final  prod- 
ucts of  digestiou  from  the  gastro-intestinal  canal. 

THE   DIGESTION   OF    THE    CARBOHYDRATES. 

The  digestion  of  the  carbohydrates  is  essentially  effected  in  the 
small  intestine  through  the  agency  of  the  amylolytic  ferment  of 
the  pancreas,  ptyalin.  and  the  inverting  ferments  maltase,  lactase, 
and  invertin,  which  are  in  part  also  furnished  by  the  pancreas,  but 
are  principally  found  in  the  enteric  juice.  In  those  animals  in 
which  ptyalin  occurs  in  the  saliva,  amylolysis  to  a  certain  degree 
also  takes  place  in  the  mouth  and  continues  in  the  stomach  until 
hydrochloric  acid  appears  in  the  free  state,  when  the  ferment  is 
rapidly  destroyed.  In  man,  however,  the  salivary  digestion  only 
plays  a  secondary  rdle.  it  is  true  that  the  end-product  of  amylo- 
lytic digestion,  viz.,  maltose,  can  probably  always  be  demonstrated 
in  the  gastric  contents,  even  after  a  few  minutes  following  the 
ingestion  of  starch  ;  but  it  must  be  borne  in  mind  that  the  trans- 
formation of  starch  into  sugar  docs  not  take  place  in  distinct 
phases,  but  that  one  molecule  of  the  substance  may  have  already 
been  changed  to  maltose,  while  another  is  as  yet  unaffected.  In 
determining  the  intensity  and  the  extent  of  amylolytic  activity  it 
i-  hence  unwarrantable  to  draw  conclusions  from  mere  qualitative 
tests,  and  it  i-  necessary  to  compare  the  amount  of  sugar  which  is 
actually  formed  with   the  amount  of  starch    thai  lias   been    ingested. 

in    the    majority   of  the   purely   carnivorous   animals,  as  has    been 

pointed  out.  the  saliva  contain-  no  digestive  ferments,  and,  in  such, 
carbohydrate  digestion  takes  place  exclusively  in  the  small  intestine. 
Through  the  action  of  the  ptyalin  of  the  pancreatic  juice  or  of  the 
saliva,  as  the  case  may  be,  the  insoluble  starch  is  firsl  transformed 
into  soluble  starch  or  amidulin  (amylodextrin),  and  ie  then  succes- 

I6fi 


166      THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

sively  decomposed  by  hydrolysis  into  erythrodextrin,  achroodextrin, 
isomaltose,  and  finally  into  maltose,  as  shown  by  the  equations  : 

(1)  (C12H  O10)54  +3H20  =3[(C12H20O10)17.C12H22Ou] 

Amiduhn.  Erythrodextrin. 

(2)  3[(C12H20O10)17.C12H22On]  +  6H20  =9[(C12H20O10)5.C12H22Ou] 

Erythrodextrin.  Achroodextrin. 

(3)  9[(C12H20O10)5.C12H22Ou]  +  45H20  ■=  54C12H22Ou  =  54C12H2„Ou 

Achroodextrin.  Isomaltose.  Maltose. 

Glycogen  is  similarly  decomposed,  and,  like  starch,  gives  rise  to 
the  formation  of  isomaltose  and  maltose.  The  celluloses,  on  the 
other  hand,  are  not  affected  by  ptyalin,  nor,  indeed,  by  any  of  the 
digestive  fluids.  As  we  shall  see,  however,  they  undergo  a  certain 
kind  of  fermentation  under  the  influence  of  various  bacteria,  and 
as  a  result  we  find  that  in  herbivorous  animals,  at  least,  only  a  frac- 
tion of  the  ingested  cellulose  reappears  in  the  feces.  Thus  far  a 
transformation  into  maltose  or  glucose  has  not  been  observed  in  the 
intestinal  tract. 

It  is  stated  that  through  the  activity  of  the  bacteria  of  the  intesti- 
nal canal,  viz.,  their  ferments,  a  certain  amount  of  starch  is  trans- 
formed into  maltose,  and  that  inversion  of  disaccharides  to  mono- 
saccharides is  likewise  brought  about  in  this  manner.  To  what 
extent  this  bacterial  action  can  be  regarded  as  representing  an  actual 
digestion  is,  however,  an  open  question.  The  presence  of  bacteria 
in  the  intestinal  canal  is  certainly  not  imperative,  as  has  been  con- 
clusively established  by  JNuttall  and  Thierfelder;  and  wre  know, 
moreover,  that  bacterial  action  extends  far  beyond  the  action  of 
digestive  ferments,  and  that  the  further  changes  that  can  thus  be 
effected  do  not  serve  a  useful  purpose.  The  normal  inversion  of 
the  disaccharides  is  unquestionably  brought  about  by  the  invertin, 
maltase,  and  lactase,  which,  as  has  been  indicated,  are  partly  fur- 
nished by  the  pancreas,  but  principally  by  the  enteric  juice.  This 
inversion  is  likewise  of  the  nature  of  a  hydrolytic  process,  and  may 
be  represented  by  the  general  equation  : 

Ci2H22On    +    H20    =    2C6H1206 

Disaccharide.  Monosaccharide. 

To  judge  from  certain  experiments  which  have  been  performed 
on  animals,  it  appears,  however,  that  amylolysis  at  least  can  also 
take  place  in  the  absence  of  the  principal  gland  by  which  the 
ptyalin  is  formed,  viz.,  the  pancreas.  For  we  find  that  following 
the  extirpation  of  this  organ  or  ligation  of  the  pancreatic  duct  dogs 
are  still  capable  of  utilizing  as  much  as  from  47  to  71  per  cent,  of 
the  starch  ingested.  As  the  dog's  saliva  contains  no  ptyalin,  the 
amylolysis  cannot  be  referable  to  a  converting  activity  of  the  salivary 
glands.  Whether  in  such  cases  the  small  amount  of  the  ferment 
which  is  also  furnished  by  the  enteric  juice  is  sufficient  to  transform 
the  ingested  starch  into  maltose  is  questionable,  and  there  is  some 
reason  for  supposing  that  the  epithelial  cells  of  the  small  intestine 


DIGESTION  OF  THE  CARBOHYDRATES.  167 

are  likewise  capable  not  only  of  causing  the  transformation  of  disac- 
charides  into  monosaccharides,  but  also  of  inverting  dextrin  to 
maltose.  It  has  been  shown  that  in  animals  with  Thiry-Vella 
fistulse  injected  solutions  of  starch  and  cane-sugar  rapidly  disappear, 
although  maltose  cannot  at  times  be  demonstrated  in  the  fluid.  In 
what  manner  this  change  is  effected  by  the  epithelial  cells  is  not 
known.  In  any  event,  however,  it  is  necessary  that  the  polysac- 
charides should  be  inverted  to  monosaccharides  before  passing  beyond 
the  mucous  membrane  of  the  gastro-intestinal  tract.  Resorption 
takes  place  primarily  through  the  specific  activity  of  the  epithelial 
lining  of  the  gastro-intestinal  mucosa.  The  monosaccharides  then 
enter  the  blood-current  and  are  carried  to  the  muscles  and  the  liver, 
where  they  are  transformed  into  glycogen  and  stored  in  a  manner 
analogous  to  the  reserve  starch  of  the  plant.  This  transformation, 
however,  as  well  as  the  subsequent  fate  of  the  sugar,  we  shall  have 
occasion  to  study  in  greater  detail  in  a  subsequent  chapter. 

Neither  the  polysaccharides  nor  the  disaccharides  when  introduced 
into  the  blood-current  directly  can  be  utilized  by  the  body  as  such, 
and  they  are  accordingly  eliminated  in  the  urine  as  foreign  matter. 

The  extent  to  which  amylolysis  can  occur  in  the  intestinal  canal 
is  remarkable,  and  far  exceeds  the  ability  of  the  liver  and  the  muscle- 
tissue  to  transform  the  corresponding  amount  of  monosaccharides 
into  glycogen.  As  a  consequence,  the  percentage  of  circulating 
sugar  rises  beyond  the  normal  and  glucosuria  results.  That  disac- 
charides may  pass  the  intestinal  mucosa  without  being  inverted  is 
possible,  but  is  certainly  of  exceptional  occurrence.  In  such  cases 
we  must  imagine  that  the  intestinal  epithelium  has  lost  its  specific 
power  as  a  barrier  to  the  passage  of  the  sugars,  as  well  as  its  ability 
to  cause  their  inversion.  As  a  result  they  pass  this  barrier  by  diffu- 
sion, and  probably  enter  both  the  blood-  and  the  lymph-current,  and 
are  then  eliminated  in  the  urine.  A  formation  of  glycogen  from  di- 
saccharides directly  is  apparently  not  possible. 

The  rapidity  with  which  resorption  takes  place  in  the  small  intes- 
tine seems  t<>  vary  with  the  character  of  the  sugar.  In  dogs  Alber- 
tini  tli us  found  that  of  100  grammes  of  glucose,  60  grammes  are 
absorbed  in  the  course  of  the  first  hour,  while  of  maltose  and  cane- 
sugar  from  70  to  80  grammes  and  of  lactose  only  20  to  40  grammes 
disappear  within  the  same  period  of  time. 

The  ingestion  of  very  large  amounts  of  disaccharides  and  mono- 
saccharides Leads  to  a  general  disturbance  of  intestinal  digestion  and 
results  in  diarrhoea.  A  corresponding  amount  of  starch,  on  the 
other  hand,  is  without  effect  in  this  respect.  This  is  nodoubtowing 
to  the  fact  that  in  the  latter  case  inversion  and  resorption  proceed 
paripa88U,  so  that  the  bacteria  have  hut  little   chance  of  setting   up 

fermentative  changes,  which  lead  to  the  formation  of  substances 
that  directly  increase  the  peristalsis  owing  to  their  irritating  prop- 
erties.    In  the  presence  of  abnormally  large  amounts  of  sugars  as 

such,  on  the  other  hand,  resorption  is  not  sufficiently  rapid,  and  in 


168       THE  PROCESSES  OF  DIGESTION  AND   RESORPTION. 

the  presence  of  the  increased  amount  of  pabulum  an  increase  of 
bacterial  fermentation  beyond  the  normal  takes  place.  As  a  result 
the  various  acid  decomposition-products  of  the  carbohydrates  such 
as  lactic  acid,  butyric  acid,  acetic  acid,  formic  acid,  succinic  acid, 
together  with  carbon  dioxide,  methane,  and  hydrogen,  are  formed  in 
increased  amounts,  and  are  responsible  for  the  resulting  pathological 
conditions. 

DIGESTION  OF  THE  ALBUMINS. 

The  digestion  of  the  albumins  takes  place  in  the  stomach  and 
in  the  small  intestines  under  the  influence  of  the  pepsin  and  the  hy- 
drochloric acid  of  the  gastric  juice,  and  the  trypsin  of  the  alkaline 
pancreatic  juice,  respectively.  We  know  that  the  presence  of  the 
former  is  not  altogether  necessary,  however,  and  that  the  pancreatic 
juice  is  in  itself  quite  sufficient  to  accomplish  the  digestion  of  the 
albumins  alone,  but  under  normal  conditions  the  gastric  juice  also 
plays  a  part.  Of  the  relative  extent  to  which  the  one  or  the  other 
enters  into  this  process  our  knowledge  is  not  complete.  We  may 
imagine  that  in  the  stomach  a  primary  dissolution  of  the  solid  con- 
stituents of  the  food  takes  place,  and  that  the  soluble  products 
which  are  thus  formed  are  further  digested  by  the  pancreatic  juice. 
This  actually  occurs  to  a  certain  extent,  but  we  further  know  that 
in  the  stomach  certain  albuminous  food-stuffs  are  decomposed,  with 
the  liberation  of  constituents  which  are  insoluble  in  the  gastric  juice, 
and  which  pass  the  pylorus  as  such  and  are  then  modified  by  the 
pancreatic  juice.  Tryptic  digestion,  moreover,  is  far  more  extensive 
than  peptic  digestion,  so  that  we  may  well  conclude  that  the  latter 
essentially  represents  a  preliminary  phase  of  digestion;  and  that 
the  digestion  proper,  viz.,  the  transformation  of  the  albumins  into 
those  final  products  which  can  be  directly  utilized  by  the  body, 
occurs  under  the  influence  of  the  trypsin  of  the  pancreatic  juice. 

For  convenience'  sake,  we  shall  study  the  action  of  the  gastric 
juice  and  of  the  pancreatic  juice  separately  upon  the  various  classes 
of  albumins,  as  the  digestive  products  which  are  formed  are  somewhat 
different  in  the  different  classes.  In  every  case  we  shall  follow  the 
fate  of  these  various  substances  to  the  final  products,  as  we  obtain 
them  artificially  in  digestive  experiments  in  vitro ;  but  we  must 
bear  in  mind  that  such  experiments  cannot  reproduce  what  actually 
takes  place  in  the  living  body,  where  resorption  is  constantly  going 
on,  and  where  the  various  digestive  processes  in  a  manner  supple- 
ment each  other,  and  conditions  overlap.  Whenever  possible  we 
shall  attempt  to  point  out  where  gastric  digestion  probably  ceases  and 
pancreatic  digestion  begins,  but  such  an  attempt  must  of  necessity  be 
more  or  less  crude. 

Digestion  of  the  Native  Albumins. 

Gastric  Digestion. — In  the  stomach  the  native  albumins,  if  in- 
troduced in  the  coagulated  state,  are  first  transformed  into  a  soluble 


DIGESTION  OF  THE  ALBUMINS.  169 

form,  and  at  the  same  time  or  immediately  following  their  dissolu- 
tion they  undergo  the  process  of  denaturization — i.  e.,  they  are  trans- 
formed into  syntonins  or  acid  albumins,  and  as  a  consequence  all 
individual  characteristics  which  previously  existed  are  lost.  This 
transformation  is  essentially  referable  to  the  hydrochloric  acid  of  the 
gastric  juice,  and  can  be  brought  about  artificially  in  the  absence  of 
pepsin.  In  such  an  event,  however,  a  higher  grade  of  acidity  and  a 
higher  temperature  are  required.  The  presence  of  the  pepsin  ob- 
viates such  a  necessity.  A  possible  explanation  of  this  phenomenon 
is  afforded  by  the  modern  doctrine  which  teaches  that  the  action  of 
enzymes   merely  consists  in  hastening  the  rapidity  of  reaction. 

On  continued  exposure  to  the  acid  gastric  juice  the  syntonins  are 
decomposed  by  hydrolysis  into  albumoses,  and  finally  into  peptones, 
and  it  is  to  be  noted  that  this  result  also  can  be  effected  by  hydro- 
chloric acid  alone ;  but  here  again  the  presence  of  the  pepsin  does 
away  with  the  necessity  of  employing  stronger  solutions  of  the  acid 
and  a  higher  temperature. 

This  decomposition  may  be  compared  to  the  inversion  of  the  poly- 
saccharides to  monosaccharides,  and  here,  as  there,  intermediary  prod- 
ucts are  formed  which  differ  not  only  from  the  syntonins,  but  also 
from  each  other.  Kuhne  and  his  school,  who  have  largely  contribu- 
ted to  our  knowledge  of  these  products,  have  suggested  their  divi- 
sion into  two  classes,  viz.,  the  primary  and  secondary  albumoses, 
according  to  their  nearer  or  more  distant  relationship  to  the  original 
albumins.  This  division  has  been  generally  accepted.  Further 
researches  have  shown  that  two  distinct  varieties  of  the  primary 
albumoses  exist,  namely,  a  proto-albumose  and  a  hetero-albumose, 
each  of  which  on  further  decomposition  is  supposed  to  give  rise  to  a 
secondary  albumose  or  deutero-albumose,  from  which  in  turn  a  pep- 
tone is  derived.  According  to  Neumeister,  the  digestion  of  the  native 
albumins  may  accordingly  be  represented  by  the  following  schema  : 

Native  albumin. 

I  . 
Syntonin. 


Proto-albumose.  Hetero-albumose. 

I  I 

Deutero-albumose.  Deutero-albumose. 

I  I 

Peptone  Peptone. 

With  the  formation  of  peptones  peptic  digestion,  according  to 
Kuhne,  comes  to  an  end.  Supposing  this  to  be  the  case,  and  bear- 
ing in   mind  that   the  albuminous  molecule  contains  certain  atomic; 

groups — the  hemi-groups — which  can    be  readily  broken  down  with 

trypsin,  with  tin-  formation  of  amido-acids,  while  others — the  anti- 
groups — are  more  resistant,  the  conclusion  suggests  itself  that  in  the 

albumoses  and    the  peptones   which   result   from  the  action  of  pep-in 

both  of  these  groups  musl  still  be  united.     Such  peptones  Kuhne 


170       THE  PROCESSES   OF  DIGESTION  AND  RESORPTION. 

has  accordingly  termed  amphopeptone.  It  has  further  been  observed 
that  during  peptic  digestion  a  certain  amount  of  an  insoluble  albu- 
minous substance  usually  remains  behind,  which  is  characterized  by 
its  great  resistance  to  further  decomposition  by  means  of  pepsin  and 
hydrochloric  acid.  This  substance  Kiihne  has  termed  anti-albumid, 
and  he  supposed  that  in  it  certain  anti-groups  were  already  isolated. 
Within  recent  years  our  conception  of  the  decomposition  of  the 
albumins  as  just  outlined  has  undergone  certain  modifications,  and 
through  the  researches  of  the  Strassburg  school,  and  notably  those 
of  Pick,  Zuntz,  and  others,  our  knowledge  of  the  products  of  diges- 
tion has  been  much  extended.  It  has  thus  been  shown  that  Kiihne' s 
views,  as  regards  the  formation  of  the  primary  albumoses  from  the 
syntonins,  were  in  the  main  correct,  and  that  proto-albumose  as 
well  as  hetero-albumose  develop  from  the  latter  simultaneously,  and 
are  not  derived  the  one  from  the  other,  as  was  once  supposed.  These 
observers  have  further  shown,  however,  that  one  deutero-albumose 
at  least  is  also  formed  at  the  same  time,  and  which,  in  contradistinc- 
tion to  the  other  two  primary  albumoses,  contains  the  carbohydrate 
group  of  the  original  albumin.  This  deutero-albumose  is  spoken  of 
as  the  deutero-albumose-B.  Whether  still  other  primary  albumoses 
exist  is  as  yet  an  open  question,  but  there  is  reason  to  suppose  that 
this  may  be  the  case,  and  it  appears,  moreover,  that  not  all  albumins 
give  rise  to  the  same  primary  albumoses,  and  that  distinct  quantita- 
tive differences  further  exist.  These  primary  albumoses  on  further 
digestion  with  pepsin  give  rise  to  secondary  albumoses.  But, 
while  according  to  Neumeister's  schema  one  deutero-albumose  only 
results  from  every  primary  albumose,  the  studies  of  Pick  clearly 
show  that  proto-albumose  yields  two  secondary  albumoses,  which 
have  been  termed  albumoses  A  and  B.  This  B-albumose,  how- 
ever, differs  from  the  primary  albumose  which  is  designated  by  the 
same  letter  in  containing  no  carbohydrate  group,  and  for  conveni- 
ence' sake  we  shall  speak  of  the  secondary  product  as  albumose-B'. 
The  hetero-albumose  similarly  yields  three  secondary  albumoses — 
that  is  A,  B',  and  C.  The  further  decomposition  of  the  primary 
albumose  B  finally  has  not  been  studied  in  detail,  and  it  may  indeed 
be  questionable  whether  it  actually  represents  one  single  substance. 
On  prolonged  digestion  with  pepsin  it  yields  a  peptone,  A,  which  is 
insoluble  in  alcohol,  and  an  albumose  which  is  manifestly  identical 
with  the  secondary  albumose  B'.  Of  the  subsequent  fate  of  the 
secondary  albumoses  we  know  little,  as  these  bodies  have  not  as 
yet  been  studied  in  this  direction.  On  prolonged  digestion  of  the 
primary  albumoses  with  pepsin  substances  are  obtained  which,  ac- 
cording to  Kiihne' s  definition,  would  correspond  to  peptones.  In 
the  mixture  of  the  secondary  albumoses,  which  Pick  obtained  from 
hetero-albumose,  peptone  was  found,  which  was  soluble  in  alcohol, 
and  which  he  termed  peptone-B.  From  proto-albumose,  on  the 
other  hand,  peptone-like  bodies  were  obtained  which  gave  an  intense 
biuret  reaction,  and  which,  of  course,  could  not  correspond  to  the 


DIGESTION  OF  THE  ALBUMINS. 


171 


peptone-A.  For  the  present  we  shall  speak  of  this  also  as  peptone  B. 
According  to  the  above  considerations,  we  may  possibly  represent 
the  process  of  peptic  digestion  by  the  following  schema,  which,  how- 
ever, is  only  provisional,  and  merely  represents  the  facts  as  just 
outlined  : 


Native  albumin 

I 
Syntonin 


Proto-albumose  Hetero-albuinose  Deutero-alburuose-B  Anti-albumid 


I  II  II 

Deutero-albuinoses      Deutero-albumoses        Deutero-albumose-B' 

I  I  I  I  I 

A  W     A  B'  C 


Proto-albumose. 
Contains  25  per  cent,  of  total  nitrogen  in 

basic  form. 
Yields   much    tyrosin,    viz.,    indol    and 

skatol. 


Peptone-B  Peptone-B  Peptone-B    Peptone-A 

Whether  or  not  the  different  albumoses  which  arc  thus  formed 
during  the  process  of  peptic  digestion  are  qualitatively  the  same, 
irrespective  of  their  origin,  we  do  not  know,  but  it  is  likely  that 
certain  differences  exist.  Quantitative  variations  also  occur  without 
doubt,  as  is  a  priori  suggested  by  the  varying  amounts  of  the  amido- 
acids  of  the  fatty  series  and  of  the  aromatic  group  which  are  con- 
tained in  the  original  albuminous  molecule,  as  also  by  the  absence 
of  a  carbohydrate  group  in  some  of  the  albumins,  the  digestion  of 
which  is  quite  analogous  to  that  of  the  native  albumins.  The 
principal  points  of  difference  between  proto-albumose  and  hetero- 
albumose  are  here  given. 

Hetero-ai.bumose. 
Contains  39  per  cent,  of  the  total  nitrogen 

in  basic  form. 
The  aromatic  group  is  present  only  to  a 

slight  extent  in  a  form  which  can  give 

rise  to  tyrosin  or  indol. 
Yields  much  leucin  and  glycocoll.  |  Yields  but  little  leucin  and  glycocoll. 

It  is  thus  manifest  that  those  albumins  which  are  especially  rich  in 
aromatic  groups  will  furnish  a  correspondingly  larger  amount  of 
proto-albumose  than  those  in  which  the  fatty  acid  radicles  arc  prin- 
cipally found,  and  which  accordingly  yield  a'  large  amount  of  hetero- 
albumose.  That  these  differences  will  further  become  manifesl  in 
the  secondary  product-  of  decomposition  i-,  of  course,  apparent. 

Of  the  character  of  the  final  products  of  peptic  digestion,  in  the 
sense  of  Ktihne,  viz.,  the  peptones,  our  knowledge  is  very  imperfect. 

While  according  to  olaer  views  amphopeptone  was'  thought  to 
represent  a  chemical  unity,  Pick  and  others  have  Bhown  thai  two 
peptones  at  least   resull  from  the  action  of  pepsin  on  albumin,  one 

of  which  is,  soluble  in  alcohol,  while  the  other  is  insoluble.  These 
bodies,  however,  are  most  likely  n,,t  units  in  themselves,  but  prob- 
ably represent  mixture-  of  other  substances,  which  for  the  most  part 
are  as  yet   unknown. 


172       THE  PROCESSES   OE  DIGESTION  AND  RESORPTION. 

Until  quite  recently  it  was  thought  that  peptic  activity  ceased 
with  the  formation  of  amphopeptone,  in  the  sense  of  Kiihne,  and 
that  other  nitrogenous  decomposition-products  beyond  those  already 
considered  were  not  formed  during  this  process.  In  the  light  of 
modern  investigations,  however,  this  view  can  no  longer  be  upheld, 
for  it  has  been  shown  that  in  a  very  early  stage  of  digestion 
already,  and  before  the  formation  of  peptones  and  the  deutero- 
albumose-C,  at  least,  a  very  considerable  portion  of  the  albuminous 
nitrogen  is  split  oif  in  the  form  of  substances  which  no  longer  give 
the  biuret  reaction.  It  is  known,  moreover,  that  the  greater  portion 
of  the  final  decomposition-products  which  result  from  the  action  of 
pepsin  do  not  consist  of  either  albumoses  or  peptones,  but  appar- 
ently of  these  same  bodies  which  do  not  give  the  biuret  reaction.  Of 
their  nature,  however,  and  their  mode  of  origin,  nothing  is  known. 

From  what  has  been  said,  it  is  thus  clear  that  the  peptic  disinte- 
gration of  the  albuminous  molecule  is  very  much  more  complicated 
than  was  formerly  supposed,  and  much  work  must  still  be  done 
before  we  can  form  a  clear  idea  of  the  entire  process. 

As  regards  the  extent  to  which  peptic  digestion  is  carried  in  the 
stomach,  our  knowledge  is  likewise  not  complete,  but  there  is  reason 
for  the  assumption  that  ordinarily  the  process  does  not  extend  beyond 
the  formation  of  the  primary  albumoses.  We  find,  as  a  matter  of 
fact,  that  while  these  appear  within  the  first  half-hour  of  digestion, 
the  secondary  albumoses,  in  experiments  in  vitro  at  least,  are  not 
formed  until  after  the  second  hour,  beyond  traces,  and  it  is  to  be 
noted  that  a  more  energetic  formation  in  fact  does  not  occur  within 
the  period  of  time  during  which  remnants  of  an  ordinary  meal  are 
usually  found  in  the  stomach.  We  are  thus  forced  to  the  conclusion 
that  if  a  more  extensive  decomposition  of  the  albumins  is  necessary 
before  resorption  can  take  place,  this  must  occur  in  the  small 
intestine  under  the  influence  of  the  pancreatic  juice.  But  we  may 
also  suppose  that  the  epithelial  lining  of  the  gastric  mucosa  can 
bring  about  the  further  transformation  of  the  primary  albumoses 
of  itself.  We  know,  as  a  matter  of  fact,  that  in  the  resorption  of 
the  albumins,  as  in  that  of  the  carbohydrates,  the  epithelial  cells 
play  an  active  part,  and  that  absorption  by  osmosis  under  normal 
conditions  can  scarcely  enter  into  consideration.  We  have  seen, 
moreover,  that  an  inversion  of  polysaccharides  to  monosaccharides 
can  thus  be  effected,  and  we  have  reason  to  suppose  that  a  digestion 
of  albumins  also  can  be  accomplished  in  the  same  manner.  It  is 
even  claimed  that  a  resorption  of  native  albumins  can  take  place  in 
the  absence  of  the  proteolytic  ferments,  and  that,  unlike  the  poly- 
saccharides, solutions  of  such  albumins  can  be  injected  directly 
into  the '  blood-current  without  causing  albuminuria.  We  have  seen 
that  the  presence  of  the  disaccharides  in  the  blood  at  once  leads  to 
their  elimination,  and  that  the  body  is  manifestly  incapable  of  caus- 
ing their  inversion  and  further  transformation  into  glycogen  when 
they  are  present  as  such.     As  the  introduction  into  the  blood-current 


DIGESTION  OF  THE  ALBUMINS.  173 

of  certain  albumins,  such  as  the  syntonins,  obtained  from  myosin, 
fibrin,  etc.,  does  not  lead  to  their  elimination  in  the  urine,  and  as  the 
blood  manifests  a  very  strong  tendency  to  maintain  its  composition 
uniform,  it  has  been  argued  that  these  albumins  are  probably  re- 
tained in  some  organ  of  the  body,  such  as  the  liver,  and  may  here 
be  transformed  into  material  which  can  be  utilized  for  purposes  of 
nutrition.  Such  an  assumption  seems  to  me  entirely  unwarrantable, 
however,  as  it  is  solely  based  upon  the  non-appearance  of  these 
albumins  in  the  urine  in  the  state  in  which  they  were  introduced. 
Egg-albumin,  it  is  true,  is  immediately  eliminated  under  such  con- 
ditions, and  it  might  be  concluded  that  the  kidneys  could  also 
eliminate  those  other  forms,  if  the  body  were  incapable  of  utiliz- 
ing them  as  such.  This,  however,  does  not  follow,  for  we  know 
that  the  introduction  of  egg-albumin  in  large  amounts  into  the 
stomach  leads  to  its  appearance  in  the  blood  as  such,  so  that  the 
conditions  here  are  reversed ;  but  it  would  be  manifestly  inadmis- 
sible to  conclude  from  this  observation  that  because  egg-albumin 
can  pass  the  epithelial  barrier,  as  such,  it  cannot  be  utilized  within 
the  body  itself  or  cannot  be  digested  in  the  stomach  and  intestines. 
That  the  introduction  of  such  albumins  which  are  normally  present 
in  the  blood  does  not  lead  to  albuminuria  is,  of  course,  not  sur- 
prising ;  but  to  conclude  that  syntonin  can  be  utilized  by  the  body 
directly  because  it  is  not  eliminated  in  the  urine  in  the  same  form 
is,  as  I  have  said,  unjustifiable. 

On  the  other  hand,  we  must  admit  that  the  resorption  of  native 
albumin  can  occur  in  the  absence  of  the  proteolytic  ferments,  and  it 
is  more  than  probable  that  during  this  resorption  the  epithelial  cells 
bring  about  a  rearrangement  of  the  atomic  groups  of  the  albu- 
minous molecule,  so  that  the  ultimate  result  is  the  same  as  though 
the  hydrolytic  decomposition  had  proceeded  further  under  the  influ- 
ence of  the  ferments  and  resorption  had  then  occurred.  Of  the 
extent  to  which  epithelial  activity  enters  into  the  process  of  diges- 
tion, however,  Ave  know  nothing,  but  it  is  likely  that  under  normal 
conditions  fermentative  digestion  prevails,  and  that  the  function  of 
the  epithelial  cells  principally  consists  in  transforming  the  albumins 
and  peptones  into  material  which  can  be  utilized  by  the  body  for 
purposes  of  nutrition.  That  this  transformation  actually  takes 
place  within  the  epithelial  cells  which  line  the  gastro-intestinal 
mucosa  is  now  established  beyond  a  doubt.  Formerly  it  was  sup- 
posed  that  this  change  occurred  within  the  liver,  especially  as 
neither  albumoses  nor  peptones  can  normally  be  found  in  the  periph- 
eral circulation.  lint  this  ha-  since  been  disproved  in  many  ways.  It 
ha-  thus  been  found  that  in  animals  which  are  killed  by  bleeding  from 
the   portal  vein  ;it  a  time  when    peptones  arc  abundantly  present    in 

the  intestines  no  peptones  can  be  found  in  the  blood.  Whenever 
peptones  are  introduced  into  the  blood  artificially,  or  whenever  they 
are  formed  beyond  the  intestinal  mucous  membrane,  as  under  certain 
pathologic  conditions,  they  are  invariably  eliminated  in  the  urine  as 


174       THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

foreign  material.  The  same  indeed  holds  good  of  the  albumoses, 
and  both  are  known  to  be  distinctly  toxic.  This  fact  in  itself  shows 
that  after  the  stage  of  denaturization  has  been  passed  a  direct  resorp- 
tion of  the  resulting  products  of  digestion  cannot  take  place  by 
simple  osmosis,  and  that  certain  changes  must  occur  in  the  gastro- 
intestinal walls  whereby  these  products  are  deprived  of  their 
toxicity.  Ludwig  and  Salvioli,  moreover,  have  shown  that  by  sub- 
stituting an  artificial  circulation  in  an  isolated  intestinal  loop  for 
the  normal,  no  peptones  or  albumoses  could  be  found  either  in  the 
blood  returning  from  the  loop  nor  after  some  time  in  the  gut  itself, 
although  an  abundant  amount  had  been  previously  introduced.  The 
same  result  may  be  demonstrated  by  finely  hashing  a  piece  of  care- 
fully cleansed  and  perfectly  fresh  intestine,  and  placing  this  in  a 
solution  of  peptone  in  the  defibrinated  blood  of  the  same  animal. 
It  can  then  be  demonstrated  that  after  a  relatively  short  time  already 
a  considerable  amount  of  the  peptones  has  disappeared,  and  is, 
moreover,  not  stored  as  such  in  the  tissue. 

While  it  is  thus  definitely  proved  that  albumoses  and  peptones 
are  transformed  into  material  which  the  body  can  utilize  for  pur- 
poses of  nutrition,  we  know  absolutely  nothing  of  the  manner  in 
which  this  transformation  is  effected,  nor  of  the  products  which  thus 
result.  According  to  some  observers,  a  further  decomposition  of  the 
albuminous  molecule  into  smaller  atomic  groups  takes  place,  but 
thus  far  no  substances  have  been  found  in  the  tissues  and  fluids  of 
the  body  in  sufficient  amount  to  favor  such  an  explanation.  On  the 
other  hand,  it  is  possible,  as  in  the  case  of  the  carbohydrates,  that 
a  polymerization  of  peptone  radicles  occurs,  and  that  albumins  again 
result  which  are  comparable  to  those  from  which  they  are  derived. 

As  I  have  stated,  there  can  be  no  doubt  that  the  epithelial  cells 
which  line  the  gastro-intestinal  tract  are  capable  of  effecting  the 
transformation  of  syntonin,  and  possibly  even  of  native  albumins 
into  forms  which  can  be  utilized  by  the  body  directly ;  but  under 
normal  conditions  it  appears  that  the  action  of  the  epithelium 
scarcely  begins  before  the  albumins  have  been  digested  by  the  fer- 
ments to  the  stage  of  albumoses.  We  have  seen  that  in  the  stomach 
the  process  of  digestion  scarcely  goes  further  than  the  formation  of 
the  primary  albumoses,  and  the  question  naturally  suggests  itself: 
Are  the  primary  albumoses  resorbed  in  the  stomach  already  or  is  it 
necessary  that  they  be  further  exposed  to  the  action  of  the  pan- 
creatic juice?  To  this  question  an  ultimate  answer  cannot  be  given, 
but  it  is  likely  that  resorption  takes  place  in  the  stomach  to  a  limited 
extent  only,  and  that  the  greater  portion  of  the  primary  albumoses 
is  further  decomposed  in  the  small  intestine,  where  the  resorption 
processes  are  most  active.  Much  work  remains  to  be  done  in  this 
direction.    . 

2.  Tryptic  Digestion  of  the  Native  Albumins. — Upon  enter- 
ing  the  small  intestine  the  acid  gastric  contents  are  rendered  alkaline, 
the  pepsin  is  destroyed,  and  tryptic  digestion  begins. 


DIGESTION  OF  THE  ALBUMINS.  175 

The  material  which  is  exposed  to  the  action  of  the  pancreatic 
juice  consists  in  part  of  the  primary  albumoses  which  were  formed 
in  the  stomach,  in  part  of  Kiihne's  anti-albumid,  and  in  part  of 
-vntonin  and  of  native  albumins,  in  soluble  or  insoluble  form,  which 
have  escaped  the  action  of  the  gastric  juice.  The  latter  are  first 
dissolved,  and  together  with  the  syntonins  transformed  into  alkaline 
albuminate.  This  result,  analogous  to  the  formation  of  the  syntonin 
in  acid  solution,  as  well  as  the  further  decomposition  of  the  alkaline 
albuminate,  is  no  doubt  primarily  referable  to  the  action  of  the 
alkalies  of  the  pancreatic  juice,  and  merely  hastened  by  the  ferment 
which  is  at  the  same  time  present.  But  unlike  the  action  of  the 
gastric  juice,  tryptic  digestion  immediately  leads  to  the  formation  of 
deutero-albumoses  without  the  intermediary  production  of  primary 
albumoses  in  the  sense  of  Kiihne.  According  to  older  views, 
amphopeptone  then  results.  Subsecpnently  the  anti-  and  the  hemi- 
groups  become  separated  with  the  formation  of  antipeptone  and 
hemipeptone,  respectively.  Hemipeptone,  hoAvever,  is  manifestly 
only  a  hypothetical  substance,  as,  in  experiments  in  vitro  at  least, 
no  substance  of  this  character  can  be  obtained.  Instead  we  find 
amido-acids,  tryptophan,  and  other  substances,  which  are  as  yet 
imperfectly  known  whenever  the  process  of  digestion  has  extended 
beyond  the  formation  of  deutero-albumoses.  In  the  living  organ- 
isms, it  is  true,  these  products  are  found  only  in  traces,  and  we 
might  hence  imagine  that  hemipeptone  is  here  resorbed  as  soon  as 
formed.  But  beyond  the  absence  of  amido-acids  as  just  stated,  we 
have  no  actual  proof  of  such  an  occurrence. 

Antipeptone,  on  the  other  hand,  viz.,  a  substance  or  substances 
which  still  give  the  biuret  reaction,  but  which,  in  contradistinction 
to  the  albumoses,  cannot  be  precipitated  by  salting  with  ammonium 
sulphate,  can  always  be  obtained  in  experiments  in  vitro.  As 
regards  the  chemical  nature  of  the  antipeptone,  however,  opinions 
differ.  Kiihne  and  Chittenden  long  ago  questioned  the  chemical 
unity  of  the  body,  and  Kutscher  has  recently  announced  that  he 
WES  able  to  isolate  the  three  known  liexon  bases,  as  also  small 
amounts  of  leucin,  tyrosin,  and  asparaginic  acid  from  the  substance. 
In  addition,  >till  other  products  of  digestion  were  found,  which 
were  not  identified,  however.  In  my  own  laboratory  I  have 
attempted  to  repeal  Kutscher's  work,  together  with  Dr.  Amberg, 
and  we  have  found  that,  as  a  matter  of  fact,  Kiihne's  antipeptone 
does  not  represent  a  chemical  unity,  but  owing  to  Kutscher's insuffi- 
cient working  directions  we  were  unable  to  confirm  his  results  in 
detail.  It  manifestly  consists  of  two  portions  however,  one  of  which 
can  be  precipitated  with  phosphotungstic  acid.  This  portion  repre- 
—*-iit  —  about  30  per  cent,  of  Kiihne's  antipeptone,  and  according  to 
Kutscher  consists  to  the  extent  of  30—31  per  cent,  of  hexon  bases. 
Siegfried's  claim  that  antipeptone  is  identical  with  his  carnic  acid, 
and  may  be  represented  by  the  formula  C10H18N8Ow  is,  in  view  of 
Kutscher's    work   and    my  own   experience,  altogether   untenable. 


176       THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

That  it  may  constitute  a  fraction  of  Kuhne's  antipeptone,  however, 
and  perhaps  even  the  greater  portion,  on  the  other  hand,  is  quite 
possible. 

We  have  seen  in  the  preceding  section  that  in  the  stomach  the 
digestion  of  the  native  albumins  scarcely  extends  beyond  the  forma- 
tion of  the  primary  albumoses.  Their  subsequent  fate,  on  exposure 
to  tryptic  digestion,  has  recently  been  studied  by  Pick.  From  his 
researches  it  appears  that  the  proto-albumose  here  apparently  yields 
the  same  deutero-albumoses  as  those  which  were  obtained  on  peptic 
digestion,  and  here  as  there  the  secondary  albumose-C  was  want- 
ing. Hetero-albumose  gives  rise  to  the  formation  of  the  deutero- 
albumoses  A  and  B',  but  curiously  enough  the  C-albumose  does  not 
appear.  The  fate  of  the  primary  deutero-albumose-B,  on  the  other 
hand,  as  also  of  the  deutero-albumose-C,  is  as  yet  unknown,  but  it 
is  likely  that  the  former  is  here  also  transformed  into  the  deutero- 
albumose-B'  and  into  peptone-A.  Especially  interesting  further  is 
the  observation  of  Pick  that  the  proto-albumose,  as  also  the  hetero- 
albumose,  rapidly  disappears,  so  that  after  twenty-four  to  thirty-six 
hours  traces  only  can  be  found  in  vitro,  while  with  peptic  digestion 
a  complete  transformation  into  secondary  albumoses  is  scarcely 
effected  even  after  several  weeks. 

Of  the  subsequent  change  which  Kuhne's  anti-albumid  under- 
goes little  is  known,  but  it  is  possible  that  the  substance  is  first 
transformed  into  anti-deutero-albumose,  and  then  contributes  to  the 
formation  of  antipeptone. 

As  regards  the  distribution  of  anti-  and  hemi-groups  in  the  orig- 
inal albuminous  molecule,  it  appears  that  both  are  present  in  about 
the.  same  proportion.  With  beginning  digestion  in  the  stomach, 
however,  this  relation  is  disturbed,  and  we  find  that  the  hetero- 
albumose  contains  rather  more  nitrogen  in  the  basic  form — 39  per 
cent. — than  the  proto-albumose — 25  per  cent.  Of  the  further  sepa- 
ration of  the  two  groups,  we  know  nothing,  and  it  is  indeed  a 
matter  of  doubt  whether  a  complete  separation  occurs  at  any  time 
preceding  the  formation  of  antipeptone,  and,  as  has  been  seen,  this 
consists  in  part  at  least  of  amido-acids,  which,  however,  are  here 
present  in  the  free  state. 

I  have  pointed  out  that  in  the  small  intestine  amido-acids  arc 
found  only  in  traces,  and  that  the  existence  of  a  hemipeptone  is 
extremely  doubtful.  The  question  hence  arises  :  In  what  form  are 
the  digestive  products  of  the  albumins  here  absorbed  ?  As  anti- 
peptone, or  its  decomposition-products,  so  far  as  we  know,  are  not 
eliminated  in  the  feces,  we  may  conclude  that  if  this  complex  of 
digestive  products  is  formed  at  all  in  the  living  body,  its  absorption 
must  take  place  in  the  intestinal  canal.  But  as  Ellinger  has  shown 
that  it  is  impossible  to  maintain  the  nitrogenous  equilibrium  by  the 
administration  of  hexon  bases  alone,  this  portion  of  the  antipep- 
tone can  scarcely  enter  into  consideration  from  the  standpoint  of 
nutrition.     Whether    or  not   those  other  bodies,    which  appear  to 


DIGESTION  OF  THE  ALBUMINS.  177 

constitute  the  greater  portion  of  the  antipeptone  and  of  the  nature 
of  which  nothing  is  as  yet  known,  can  serve  as  food-stuffs  in  the 
narrower  sense  of  the  term,  remains  to  be  seen.  Under  such  con- 
ditions it  is  perhaps  wiser  to  conclude  that  the  deutero-albumoses 
represent  those  products  of  tryptic  digestion  which  actually  play 
a  role  in  the  process  of  nutrition,  and  to  suppose  that  a  formation 
of  antipeptone  is  essentially  a  process  which  takes  place  in  vitro, 
and  normally  occurs  only  to  a  slight  extent  in  the  living  organism. 
This,  however,  is  a  mere  assumption  and  lacks  experimental  proof. 

In  the  account  of  the  process  of  albuminous  digestion,  as  out- 
lined in  the  foregoing  pages,  it  has  in  a  manner  been  assumed  that  the 
digestive  products  which  are  thus  formed  are  identical,  irrespective 
of  their  origin.  Strictly  speaking,  this  is  not  the  case,  however,  as  we 
know  as  a  matter  of  fact  that  the  distribution  of  the  nitrogen  in  the 
original  albuminous  molecule  differs  in  the  different  albumins.  The 
amount  of  hexon  bases,  moreover,  which  may  be  obtained  from  the 
various  albumins  is  not  constant,  and  we  have  reason  to  think  that 
the  number  of  carbohydrate  groups  also  is  more  or  less  variable. 
Such  differences,  it  is  true,  have  thus  far  been  mainly  established 
for  the  original  substances,  but  it  is,  of  course,  manifest  that  the 
corresponding  products  of  digestion  cannot  be  the  same.  With  the 
usual  analytical  methods,  however,  these  differences  are  scarcely 
apparent,  and  ammonium  sulphate,  which  is  now  so  extensively  uti- 
lized in  separating  the  various  albumins  from  each  other,  apparently 
acts  in  the  same  manner  with  these  products,  no  matter  what  their 
origin  may  have  been.  In  the  accompanying  tables  I  have  collected 
various  data  from  the  literature  to  show  the  difference  in  the  distribu- 
tion of  the  nitrogen  and  the  corresponding  amount  of  arginin  which 
can  be  obtained  from  some  of  the  more  important  albumins. 

Amido-    Diamino-  Monamino- 
nitrogen.  nitrogen,    nitrogen. 

Crystallized  egg-albumin  (Hansmann) 8.53  21.33  67.80 

Crystallized  serum-albumin  (Hausmann)  ■    .    .    .  6.34 

Serum-globulin  (Hausmann) 8.90  24.95  68.28 

Casein  [Hausmann) 13.38  11.71  75.98 

Gelatin  (Hausmann)      1.61  35.83  62.56 

Proto-albnmoae  of  fibrin  (Pick) 7.14  25.42  68.17 

Hetero-albumose  of  fibrin  (Pick) 6.45  38.93  57.40 

Arginin. 

Keratin 2.25  per  eent. 

Clutin 2.6     ''       " 

Conglutin 2.75  •'      " 

Albumin  (yolk) 2.3     "       " 

Albumin  (white  of  egg) 0.8    "      " 

Dried  Mood-serum 0.7     "       " 

Casein 0.25  "      " 

To  distinguish  the  different  albumoses  which  arc  derived  from 
the  true  albumins,  including  those  which  result  on  the  decomposition 
of  the   proteida    from  the  albuminoid  albumoses,  Chittenden  has  in- 

12 


178       THE  PROCESSES  OF  DIGESTION  AND  RESORPTION 

troduced  the  generic  term  proteoses,  and  according  to  their  individual 
origin  divides  the  proteoses  into  globulinoses,  vitelloses,  fibrinoses, 
myosinoses,  seroses,  etc.  Their  stages  of  digestion  are  further  indi- 
cated by  the  prefixes  proto-,  hetero-,  and  deutero,  so  that  we  speak 
of  a  proto-  and  a  hetero-vitellose,  of  deutero-caseoses,  etc. 

Digestion  of  the  Proteids. 

The  digestion  of  the  proteids,  or  of  the  nucleo-albumins,  the 
glucoproteids,  and  the  haemoglobins  at  least,  like  that  of  the  native 
albumins,  begins  in  the  stomach.  Here  the  separation  of  the  non- 
albuminous  pairling  is  first  effected,  and  is  then  followed  by  the 
digestion  of  the  liberated  albumins.  This  digestion  is  in  all  respects 
analogous  to  that  of  the  native  albumins  proper.  Syntonins  are  first 
formed,  then  primary  albumoses,  subsequently  secondary  albumoses, 
and  finally  peptones — i.  e.,  bodies  which  still  give  the  biuret  reac- 
tion, but  which  in  contradistinction  to  the  albumoses  are  not  pre- 
cipitated by  salting  with  ammonium  sulphate.  The  individual 
products  which  thus  result  from  the  proteids  have  not  as  yet  been 
studied  with  the  same  care  as  those  which  are  derived  from  the 
native  albumins,  but  it  is  likely  that  here  also  Kuhne's  schema  of 
digestion  does  not  apply  in  its  original  form.  Individual  differ- 
ences also  no  doubt  exist  between  the  various  digestive  products 
according  to  their  origin,  but  of  these  also  we  know  but  little. 

Of  special  interest  are  the  earlier  phases  of  digestion  of  the 
casein  of  milk.  This  normally  exists  in  the  milk  in  solution  as  a 
neutral  calcium  salt.  In  the  stomach  a  transformation  into  the 
corresponding  acid  salt  is  then  first  effected  by  the  hydrochloric 
acid  of  the  gastric  juice,  and  followed  by  the  action  of  the  chymosin. 
According  to  Hammarsten,  this  effects  a  partial  decomposition  of  the 
soluble  acid  salt  with  the  formation  of  calcium-paracasein,  and  a 
small  amount  of  an  albumose-like  posset  albumin.  The  paracasein 
is  then  precipitated  and  decomposed,  with  the  formation  of  the  cor- 
responding paranuclein  and  the  albuminous  pairling. 

Of  the  fate  of  the  non-albuminous  components  of  the  proteids 
but  little  is  known.  The  paranuclein  of  casein,  it  is  stated,  undergoes 
solution  on  continued  digestion  in  vitro,  but  is  at  the  same  time  de- 
composed with  the  formation  of  a  small  amount  of  orthophosphoric 
acid  and  an  organic  acid,  which  likewise  contains  phosphorus.  Of 
this,  however,  nothing  further  is  known  (see  also  page  77). 

The  nucleins  proper  are  not  digested  in  the  stomach  and  remain 
undissolved. 

Under  the  influence  of  the  pancreatic  juice  casein  is  digested  in 
very  much  the  same  manner  as  with  the  gastric  juice,  but  in  this 
case  the  transformation  into  paracasein  is  brought  about  through 
the  influence  of  the  chymosin  of  the  pancreas  in  an  alkaline  medium. 
Caseoses  then  result  as  with  the  common  native  albumins,  and  finally 
peptone  is  formed. 


DIGESTION   OF  THE  ALBUMINS.  179 

Aside  from  its  proteid  character,  casein  differs  from  the  common 
albumins  in  one  very  important  particular,  namely,  in  the  exceed- 
ingly small  number  of  hexon  groups  which  the  substance  apparently 
contains.  In  its  antipeptone,  therefore,  which  has  not  as  yet  been 
studied  in  this  direction,  however,  we  can  hence  not  expect  these 
bodies  beyond  traces. 

The  glucoproteids  and  the  haemoglobins  are  decomposed  as  in  the 
case  of  the  gastric  juice,  and  the  albuminous  components  further 
digested  like  the  native  albumins.  The  individual  products,  how- 
ever, which  are  thus  formed  have  not  as  yet  been  studied  in  detail. 

The  true  nucleins,  which,  as  we  have  seen,  escape  gastric  diges- 
tion and  which  do  not  undergo  solution  in  the  stomach,  are  dissolved 
by  the  pancreatic  juice  and  are  decomposed  with  the  liberation  of 
the  contained  nucleinic  acids  and  the  albuminous  radicles.  The 
latter  are  further  digested  in  the  usual  manner.  The  paranucleins 
similarly  undergo  dissolution,  and  are  probably  decomposed  as 
already  indicated.  Of  the  subsequent  fate  of  the  non-albuminous 
pairlings  of  the  proteids  in  general,  however,  but  little  is  known. 

Digestion   of  the   Albuminoids. 

The  only  albuminoids  which  are  digested  in  the  stomach  in  the 
case  of  the  higher  vertebrate  animals  are  collagen  and  elastin. 
Both  then  give  rise  to  protogelatose  and  proto-elastose,  respectively, 
while  curiously  enough  hetero-albumoses  are  not  formed.  The  cor- 
responding deutero-albumoses  then  result.  But  while  the  deutero- 
gelatose  subsequently  gives  rise  to  the  formation  of  peptone — the 
BO-called  glutin-peptone — a  similar  transformation  of  the  deutero- 
elastose  apparently  does  not  occur. 

In  the  small  intestine,  under  the  influence  of  the  pancreatic 
juice,  collagen  and  elastin  can  also  be  digested,  and  it  is  note- 
worthy that  the  transformation  of  the  gelatins  into  glutin-peptone 
is  apparently  more  readily  effected  than  that  of  any  other  albumin- 
ous substance.  Unlike  the  gastric  juice,  however,  the  pancreatic 
secretion  i-  in  itself  not  capable  of  transforming  the  native  collagen 
into  gelatin.  This  change  must  hence  be  first  effected  artificially 
or  in  the  stomach  before  its  further  digestion  can  occur.  The 
peptonization  of  elastin  in  the  pancreatic  juice  likewise  ceases  with 
the  formation  of  deutero-elastose,  while  gelatin  is  transformed  /'// 
vitro,  &\  least,  into  glutin-peptone.  According  to  Chittenden,  this 
i-  not  further  decomposed  by  the  trypsin,  and  amido-acids  are  hence 
not  formed.  This  i-  rather  remarkable,  as  on  hydrolytic  decom- 
position with  mineral  acids  gelatin  yields  lencin,  asparaginic  acid, 
glutarainic acid, and  considerable  amount-  ofglycocoll.    The  existence 

of  aromatic  groups    in    the  original    molecule,  on   the  other  hand,  is 
very   doubtful,    and    as  a    matter  of    fid    it    i-    impossible   to  obtain 

either  tyrosin,  or  indol,  or  skatol,  from  the  substance,  even  on  bac- 
terial  decomposition.     Hexon  groups,  however,  are  largely  present. 


180       THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

The  fact  that  neither  elastin  nor  collagen  (gelatin)  gives  rise  to 
the  formation  of  hetero-albumoses  is  of  special  interest  in  view  of 
the  fact  that  both  substances  cannot  be  regarded  as  true  food-stuffs, 
viz.,  thev  are  unable  to  maintain  the  nitrogenous  equilibrium  of  the 
higher  animals  when  exclusively  used.  Whether  or  not  this  is  due 
tothe  absence  of  the  aromatic  group  in  the  gelatin,  and  its  presence 
only  in  small  amounts  in  elastin,  is  not  decided.  The  hexon  bases 
are  manifestly  of  no  moment  in  this  connection,  as  gelatin,  at  least, 
yields  rather  more  arginin  than  any  of  the  common  albuminous  food- 
stuffs, and,  as  I  have  pointed  out,  Ellinger  has  shown  that  the 
hexon  bases  alone  are  likewise  not  capable  of  maintaining  nitro- 
genous  equilibrium.  Future  researches,  no  doubt,  will  explain  this 
essential  difference  between  the  albumins  proper,  including  the 
proteids  and  the  albuminoids. 

A  digestion  of  other  albuminoids,  notably  of  the  keratins,  does 
not  take  place  in  the  small  intestine  of  the  higher  animals,  while 
some  of  the  invertebrates,  such  as  the  common  house  moth,  are 
manifestly  capable  of  utilizing  these  also  for  purposes  of  nutrition. 
In  contradistinction  to  collagen  and  elastin,  the  keratins  yield  a 
relatively  large  amount  of  tyrosin,  in  addition  to  leucin,  aspara- 
ginic acid,  and  glutaminic  acid,  on  hydrolytic  decomposition. 

DIGESTION  OF  THE  FATS. 

Notwithstanding  innumerable  researches  in  this  direction,  our 
knowledge  of  the  digestion  of  tats  and  their  subsequent  absorption 
is  still  imperfect.  This  is  true  more  especially  of  the  role  which 
the  pancreatic  juice  and  the  bile  play  in  the  process.  That  both 
secretions  are  actively  concerned  in  the  digestion  of  the  tats  can- 
not be  doubted.  Minkowski  and  Abelmann  have  thus  shown  that 
following  extirpation  of  the  pancreas  in  dogs  the  absorption  of  fats 
ceases  altogether,  if  we  except  the  fat  of  butter,  of  which  from  28  to 
53  per  cent,  can  still  be  utilized.  Other  observers,  it  is  true, 
obtained  results  which  differ  somewhat  from  those  of  Minkowski ; 
but  in  all  cases  it  could  at  least  be  demonstrated  that  in  the  absence 
of  the  pancreatice  juice  the  absorption  of  fats  is  impeded.  In  other 
experiments  in  which  the  bile  was  prevented  from  entering  the 
intestinal  tract  it  was  similarly  demonstrated  that  only  one-seventh 
to  one-half  of  the  fat  was  resorbed.  while  the  remainder,  princi- 
pally in  the  form  of  fatty  acids,  appeared  in  the  feces.  This  is  the 
more  remarkable,  since  Munk  has  shown  that  the  fatty  acids  can 
be  absorbed  as  such,  and  are  retransformed  into  neutral  tats  in  the 
intestinal  mucous  membrane. 

While  the  importance  of  the  pancreatic  juice  and  the  bile  in  the 
digestion  of  the  fats  is  thus  manifest,  we  have  no  clear  conception 
of  the  manner  in  which  their  presence  favors  their  resorption.  It  is 
generally  stated  that  this  is  primarly  dependent  upon  a  previous 
emulsitication,  which  is  supposedly  effected  through   the   activity  of 


DIGESTION  OF  THE  FATS.  181 

the  steapsin  of  the  pancreatic  juice.  This,  us  we  have  seen,  brings 
about  a  partial  decomposition  of  the  neutral  fat,  and  it  is  thought 
that  the  resulting  fatty  acids  combine  with  the  alkalies  of  the  pan- 
creatic juice  and  the  bile  to  form  soaps,  and  that  these  in  turn 
emulsify  the  neutral  fats.  We  might  hence  conclude  that  the  pres- 
ence of  the  alkali  in  these  secretions  is  the  essential  factor  which 
renders  the  absorption  of  the  fats  possible.  I  have  shown  that  the 
succus  entericus  is  manifestly  incapable  of  furnishing  this  in  suffi- 
cient amount,  as  large  quantities  of  the  fatty  acids  appear  in  the 
feces  as  such  when  either  the  bile  or  the  pancreatic  juice  is  pre- 
vented from  entering  the  intestinal  canal.  It  has  been  found,  on 
the  other  hand,  that  even  in  the  absence  of  the  pancreas  the  absorp- 
tion of  fats  may  be  fairly  normal  providing  that  fresh  pancreas, 
finely  hashed,  is  given  the  animal  together  with  the  fatty  food. 
Kiihne,  moreover,  has  demonstrated  that  pancreatic  juice,  even  after 
having  been  rendered  feebly  acid,  is  still  capable  of  emulsifying 
fats.  He  also  pointed  out  long  ago  that  while  the  secretion  from 
permanent  pancreatic  fistula?  can  bring  about  the  emulsification 
of  fats,  the  juice  obtained  from  temporary  fistulse  is  much  more 
potent  in  this  respect.  He  accordingly  concluded  that  this  property 
is  essentially  referable  to  albumins  which  are  present  in  solution, 
and  he  showed,  moreover,  that  these  are  capable  of  bringing  about 
the  emulsification  of  fats  even  in  feebly  acid  solution.  This  opin- 
ion is  shared  by  Minkowski  and  others,  and  the  fact  that  the  fat  of 
milk  can  be  resorbed  much  more  readily  than  other  fats  is  now 
generally  explained  upon  this  basis.  However  this  may  be,  the 
fact  remains  that  resorption  of  fats  can  only  proceed  in  a  normal 
manner  if  emulsification  has  previously  taken  place. 

At  present  there  is  a  tendency  among  physiologists  to  assume  that 
the  digestion  of  the  fats  presupposes  their  decomposition  into  fatty 
acids  and  glycerin.  The  former,  in  the  form  of  soaps,  are  then 
supposedly  absorbed  and  reconstructed  into  neutral  fats  in  the  epi- 
thelial cells,  which  possibly  obtain  the  requisite  amount  of  glycerin 
from  the  intestinal  lymph-glands.  It  must  be  admitted  that  this 
view  has  much  in  its  favor,  but  it  cannot  as  yet  be  regarded  as  an 
established  fact. 

Of  the  manner  in  which  resorption  occurs,  we  now  know  that, 
contrary  to  the  former  supposition,  according  to  which  the  leucocytes 
play  an  active  part  in  this  process,  the  epithelial  cells  are  of  prime 
importance,  and  it  seems  that  even  though  the  neutral  fats  may  be 
absorbed  directly,  a  synthesis  of  fats  from  fatty  acids  or  their  soaps 
can  here  also  take  place.  This  indeed  is  the  prevailing  idea  at  the 
present  time,  and,  as  I  have  said,  the  glycerin  which  is  necessary 
to  effect  this  synthesis  is  in  all  probability  derived  from  the  lymph- 
glands  of  the  intestinal  tract,  but  it  is  also  possible  that  it  may  be 
formed  in  the  cells  themselves  or  may  be  absorbed  together  with 
the  soaps. 

It  i-  stated  that  the  bile  assists   in   the   resorption  of  fat  from   the 


182      THE  PROCESSES  OF  DIGESTION  AND  RESORPTION. 

intestinal  tract,  but  of  the  manner  of  its  action  in  this  respect  we 
know  little  that  is  definite.  After  resorption  from  the  intestinal 
canal  the  fats  are  transferred  from  the  epithelial  cells  to  the  lymph- 
vessels,  and  subsequently  reach  the  general  circulation  through  the 
thoracic  duct. 

The  lecithins,  like  the  fats,  are  decomposed  by  steapsin  into  their 
components,  viz.,  into  glycerin-phosphoric  acid,  the  corresponding 
fatty  acids,  and  cholin.  The  former  is  then  absorbed,  and  appears 
in  part  at  least  in  the  urine  as  such.  The  fatty  acids  after  saponifi- 
cation are  then  similarly  absorbed  and  reconstructed  into  neutral 
fats,  while  cholin  is  decomposed  by  the  bacteria  which  are  present 
in  the  intestines,  with  the  formation  of  carbon  dioxide,  methane, 
and  ammonia. 


CHAPTEK    IX. 

ANALYSIS  OF  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

To  demonstrate  the  formation  of  the  various  products  of  albu- 
minous digestion  and  to  separate  the  individual  substances  from  each 
other,  the  following  plan  of  work  may  be  adopted  : 

THE  PRODUCTS  OF  PEPTIC  DIGESTION. 

One  hundred  grammes  of  moist  fibrin  that  has  been  thoroughly 
washed  in  running  water  are  placed  in  1000  c.c.  of  an  0.3  per  cent, 
solution  of  hydrochloric  acid,  to  which  a  few  grammes  of  pepsin 
have  been  added.  The  mixture  is  kept  at  a  temperature  of  40°  C, 
and  may  be  examined  for  the  primary  products  of  digestion  after 
about  two  hours,  while  the  secondary  products  can  only  be  demon- 
strated after  a  longer  period  of  time.  From  time  to  time  it  is 
then  necessary  to  ascertain  whether  free  hydrochloric  acid  is  still 
present,  and  to  add  an  additional  amount  whenever  it  is  wanting, 
so  that  the  acidity  referable  to  free  acid  remains  about  the  same 
during  the  entire  period  of  digestion.  The  occasional  addition  of 
a  little  pepsin  is  also  advisable.  The  first  specimen  for  examina- 
tion is  taken  after  two  hours.  The  liquid  is  filtered  and  neutral- 
ized with  a  dilute  solution  of  sodium  hydrate.  The  precipitate 
which  thus  forms  consists  of  syntonin  and  is  filtered  off.  The  fil- 
trate is  rendered  feebly  acid  with  very  dilute  acetic  acid,  treated 
with  an  equal  volume  of  a  saturated  solution  of  common  salt,  and 
boiled.  Any  native  coagulable  albumin  that  may  be  present  is 
thus  precipitated  and  is  filtered  off  on  cooling.  The  solution  is 
again  made  neutral  and  treated  with  an  equal  volume  of  a  satu- 
rated solution  of  ammonium  sulphate.  In  this  manner  the  primary 
albumoses  of  Kiihne  are  precipitated,  and  are  filtered  off  after  stand- 
ing for  about  one-half  hour.  To  separate  the  proto-albumose  from 
the  hetero-albumose,  the  precipitate  is  dissolved  in  hot  water  and 
treated  with  an  equal  volume  of  95  per  cent,  alcohol.  On  standing, 
the  hetero-albumose  separates  out,  while  the  proto-albumose  is  found 
in  tin;  aleoholic  filtrate.  It  is  purified  by  repeated  solution  in  hot 
water  and  precipitation  with  alcohol.  To  isolate  the  proto-albumose, 
the  alcoholic  filtrate  is  evaporated  to  dryness  on  a  water-bath,  or  the 
alcohol  h  distilled  off  in  the  vacuum.  The  remaining  material  is 
repeatedly  dissolved  in  water  ami  treated  with  alcohol  until  the 
alcoholic  solution  remains  clear  on  standing.  To  this  end,  it  is 
u-ually  necessary  to  repeat  the  solution  in  water  and  the  treatment 
with  alcohol  five  or  six  times. 

183 


184  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

The  further  steps  in  the  digestion  of  fibrin  may  be  studied  in  a 
specimen  which  has  been  kept  at  a  temperature  of  40°  C.  for  two 
to  three  weeks.  Syntonin  or  native  soluble  albumin  that  may  still 
be  present,  as  well  as  the  primary  albumoses,  are  removed  as  just 
described.  The  neutral  solution  is  then  treated  with  one-half  its 
volume  of  a  saturated  solution  of  ammonium  sulphate.  In  this 
manner  a  two-thirds  saturation  of  the  solution  is  effected,  and  on 
standing  the  deutero-albumose-A  separates  out.  This  is  filtered 
off,  and  the  solution  saturated  with  ammonium  sulphate  in  sub- 
stance. As  a  result  the  deutero-albumose-B'  is  thrown  down,  and 
on  acidifying  the  filtrate  with  one-tenth  its  volume  of  a  solution 
of  sulphuric  acid  that  has  been  saturated  with  ammonium  sul- 
phate, aud  of  which  10  c.c  correspond  in  strength  to  17  c.c.  of 
a  one-tenth  normal  solution  of  sodium  hydrate,  the  deutero-albu- 
mose-C  finally  separates  out  on  standing.  The  resulting  filtrate  is 
then  free  from  albumoses,  and  should  contain  the  amphopeptone  of 
Kuhne.  But,  as  I  have  indicated,  two  additional  fractions  can  be 
obtained  from  the  final  solution.  To  this  end,  a  solution  of  iodo- 
potassic  iodide,  containing  two  parts  of  the  iodide  to  one  part  of 
iodine,  which  has  been  saturated  with  ammonium  sulphate,  is  added 
until  precipitation  is  complete.  The  material  which  is  thus  thrown 
down  is  placed  in  96  per  cent,  alcohol.  Peptone-B  then  passes  into 
solution,  while  peptone-A  remains  undissolved.  This  portion  is  dis- 
solved in  a  little  warm  water,  the  solution  saturated  with  ammonium 
sulphate,  and  reprecipitated  with  the  iodine  solution.  The  peptone 
is  then  redissolved  in  warm  water,  reprecipitated  with  alcohol,  and 
freed  from  any  remaining  iodine  by  shaking  with  ether.  Peptone-B, 
on  the  other  hand,  is  obtained  by  evaporating  its  alcoholic  solution 
to  dryness,  when  the  residue  is  dissolved  in  water  and  freed  from 
iodine  by  shaking  with  ether. 

Pick's  deutero-albumose-B,  which,  in  contradistinction  to  the 
B'-albumose,  is  said  to  contain  carbohydrate  groups,  has  not  as  yet 
been  accounted  for  in  the  above  analytical  schema.  This  is  owing 
to  the  fact  that  Pick  has  not  indicated  the  exact  manner  in  which 
the  substance  can  be  isolated.  In  his  latest  publication  he  merely 
states  that  on  careful  purification  of  the  deutero-albumoses  which 
can  be  obtained  from  Witte's  peptone  (this  is  largely  a  mixture 
of  albumoses  derived  from  fibrin)  it  was  noted  that  the  deutero- 
albumose-B  showed  in  gradually  increasing  degree  the  existence  of 
carbohydrate  radicles,  while  A  and  C  in  pure  form  were  free  from 
these  groups.  But,  as  we  have  seen,  both  the  proto-  and  the  hetero- 
albumose  on  further  digestion  yield  a  deutero-albumose-B7  which 
manifestly  contains  no  carbohydrate  groups.  It  is  possible  that  the 
B-albumose  is  hence  precipitated  together  with  the  B'-albumose ;  but 
if  it  is  found  in  this  fraction,  it  should  be  possible  to  isolate  the 
substance  in  the  earlier  stages  of  digestion  already,  as  it  is  stated 
that  its  formation  coincides  in  point  of  time  with  that  of  Kiihne's 
primary  albumoses. 


THE  PRODUCTS  OF  TBYPTIC  DIGESTION.  185 

The  general  reactions  of  the  various  albumoses  and  the  two  pep- 
tone fractions  which  can  thus  be  obtained  from  fibrin  are  shown  in 
the  accompanying  table  (pages  186  and  187).  But  while  the  albu- 
moses, of  whatever  origin,  apparently  behave  toward  ammonium 
sulphate  in  the  same  manner,  some  of  these  at  least  ditfer  from  the 
fibrinoses  in  other  respects.  The  deviations,  however,  are  on  the 
whole  but  slight,  and  may  well  be  disregarded  at  this  place. 

THE   PRODUCTS    OF   TRYPTIC   DIGESTION. 

One  hundred  grammes  of  moist  fibrin,  as  in  the  above  experi- 
ments, are  placed  in  a  liter  of  an  0.25  per  cent,  solution  of  sodium 
carbonate,  to  which  a  few  grammes  of  commercial  pancreatin  have 
been  added.  Putrefaction  is  guarded  against  by  the  addition  of 
chloroform  and  thymol.  The  mixture  is  kept  at  a  temperature  of 
40°  C,  and  can  be  examined  after  twenty-four  to  thirty-six  hours. 
For  the  preparation  of  antipeptone,  however,  in  amounts  which 
oan  be  utilized  to  demonstrate  the  presence  of  the  hexon  bases,  it 
is  necessary  to  take  a  much  larger  quantity  of  fibrin  and  to  extend 
the  period  of  digestion  over  several  weeks.  From  1410  grammes 
Kutscher  claims  to  have  obtainetl  as  much  as  200  grammes,  but  I 
have  personally  not  been  so  successful. 

The  filtered  fluid  is  first  neutralized  with  dilute  sulphuric  acid, 
which  causes  the  separation  of  any  alkaline  albuminate  that  may  be 
present.  Coagulable  albumins  are  removed  by  acidifying  the  solu- 
tion with  acetic  acid  and  boiling.  The  solution  is  then  treated  with 
one  and  one-half  times  its  volume  of  a  saturated  solution  of  ammo- 
nium sulphate.  On  standing,  the  deutero-albumose-A  separates  out. 
On  complete  saturation  with  the  salt  in  substance  the  deutero- 
alburnose-B'  is  obtained ;  a  C-albumose  is  not  formed  on  tryptic 
digestion.  On  acidifying  with  sulphuric  acid,  however,  as  in  the 
study  of  peptic  digestion,  it  may  happen  that  a  turbidity  appears, 
which  is  probably  due  to  the  presence  of  Neumeister's  antideutero- 
albumose.  The  final  filtrate  then  contains  the  common  amido- 
a«'ids,  antipeptone,  tryptophan,  and  probably  other  substances  also 
which  are  as  yet  but  imperfectly  known. 

Leucin. — Aside  from  its  formation  during  the  process  of  pancreatic 
digestion  or  on  artificial  decomposition  of  albumin  with  dilute  mineral 
acids  and  alkalies,  leucin  has  been  demonstrated  in  the  spleen,  in  the 
Lymph-glands,  in  the  thyroid,  the  kidneys,  the  liver,  and  the  brain, 
though  mostly  under  pathological  conditions,  when  it  may  also 
appear  in  the  urine.  It  is  further  found  in  sheep's  wool,  in  decom- 
posing epithelial  structures,  as  in  the  desquamated  material  which  is 
found  between  the  toes,  etc  Its  presence  in  the  intestine  may  also 
be  due  to  the  action  of  bacteria  upon  the  albuminous  products  of 
digestion. 

Jn  pure  form  leucin  crystallizes  in    extremely  thin  white  lustrous 

platelets;  but  more  commonly  it  i-  seen  in  the  form  of  spherules  of 


186 


THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 


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188  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

variable  size,  which  closely  resemble  globules  of  fat.  In  these,  con- 
centric striatums,  as  well  as  very  fine  radiating  lines,  can  at  times  be 
made  out  on  careful  examination. 

Several  leucins  apparently  exist.  One  form,  which  can  be  pro- 
duced synthetically  from  hydrocyanic  acid  and  ammonium-iso- 
valerianic  aldehyde,  is  optically  inactive.  The  common  leucin, 
on  the  other  hand,  which  is  formed  during  tryptic  digestion  or  on 
decomposition  of  the  native  albumins  with  hydrochloric  acid,  is 
dextrorotatory.  A  third  form  results  from  the  action  of  Penicilium 
glaucum  upon  the  inactive  substance,  and  is  said  to  be  lsevorotatory. 
According  to  Cohn,  moreover,  several  isomeric,  optically  active 
leucins  exist.  The  common  form  is  easily  soluble  in  water,  in 
alkalies  and  acids,  as  also  in  hot  alcohol ;  in  ether  it  is  insoluble. 
It  combines  with  acids,  alkalies,  and  the  oxides  of  some  of  the  heavy 
metals  to  form  salts.  On  boiling  a  solution  of  leucin  with  subacetate 
of  lead  the  corresponding  compound  of  lead  oxide  can  thus  be 
obtained  if  ammonia  is  carefully  added  to  the  cooled  solution.  A 
copper  salt  is  similarly  formed  if  leucin  in  aqueous  solution  and  con- 
taining a  small  amount  of  alkali  is  treated  with  a  solution  of  cupric 
sulphate,  care  being  taken  not  to  add  an  excess.  On  standing,  the 
compound  separates  out  in  the  form  of  clusters  of  blue  needles,  which 
are  characterized  by  their  pronounced  insolubility. 

When  carefully  heated  to  a  temperature  of  170°  C.  leucin  melts 
and  sublimes  in  the  form  of  white  flakes,  which  are  deposited  on  the 
cooler  portion  of  the  tube.  At  the  same  time  the  odor  of  amylamin 
develops. 

On  evaporating  a  small  amount  of  leucin  upon  platinum  foil  with 
nitric  acid  a  colorless  residue  is  formed.  If  to  this  a  drop  of  sodium 
hydrate  solution  is  added  and  heat  is  carefully  applied,  a  yellowish 
or  brownish  color  develops,  and  on  further  heating  an  oil-like  droplet 
is  obtained,  which  rolls  about  upon  the  platinum  without  adhering 
(Scherer's  test). 

On  decomposition  with  an  alkali  or  during  the  process  of  putre- 
faction leucin  yields  ammonia  and  valerianic  acid.  On  oxidation 
leucinic  acid  results. 

As  has  been  indicated,  leucin  is  an  amido-capronic  acid  of  the 
formula  (CH3)2.CH.CH2.CH(NH2).COOH,  and  may  hence  also  be 
regarded  as  a-amido-isobutyl-acetic  acid. 

Tyrosin. — Tyrosln  can  be  obtained  on  tryptic  digestion  from  all 
those  albumins  in  which  aromatic  groups  exist.  Collagen,  in  which 
this  is  absent,  accordingly  yields  no  tyrosin,  and  very  small  amounts 
only  are  obtained  from  elastin.  In  the  animal  body  it  is  practically 
found  as  such  only  under  pathological  conditions  if  Ave  disregard  the 
minute  quantity  which  is  formed  in  the  intestinal  canal.  Like 
leucin,  it  is  also  formed  during  the  process  of  albuminous  putrefaction, 
and  can  be  obtained  artificially  by  decomposing  albuminous  sub- 
stances with  dilute  mineral  acids  or  alkalies. 

While  impure  tyrosin  may  occur  in  the  form  of  spherules  similar 


THE  PRODUCTS  OF  TRYPT1C  DIGESTION.  18$ 

to  those  of  leucin,  the  pure  substance  crystallizes  in  delicate,  silky 
needles,  which  are  often  grouped  in  sheaves  and  rosettes.  Accord- 
ing to  its  mode  of  formation,  the  substance  is  optically  inactive,  as 
when  formed  synthetically  or  by  decomposition  with  baryta-water, 
or  it  is  lsevorotatory  when  derived  from  albumins  on  boiling  with 
acids.  In  cold  water  it  is  only  slightly  soluble,  while  in  boiling 
water  it  dissolves  in  the  proportion  of  1  to  154.  Its  solubility  is 
increased  in  the  presence  of  alkalies  or  mineral  acids.  In  alcohol 
and  ether  it  is  insoluble. 

Tvrosin  may  be  regarded  as  para-oxy-phenvl-propionic  acid,  and 
has  the  formula  Ct;H4(OH).CH2.CH(NH2).COOH.  It  may  be  formed 
synthetically  from  ethylene  oxide  and  para-am ido-benzoic  acid,  and 
can  also  be  obtained  from  para-amido-phenyl  alanin  and  para-nitro- 
phenyl  alanin.  On  bacterial  decomposition  it  yields  hydroparacu- 
maric  acid  (para-oxy-phenvl-propionic  acid),  which  can  be  further 
transformed  into  para-oxy-phenyl-acetic  acid,  and  this  into  para- 
cresol,  as  has  been  shown  (page  89.)  On  fusion  with  caustic  alkali, 
on  the  other  hand,  it  gives  rise  to  the  formation  of  para-oxy-benzoic 
acid,  acetic  acid,  and  ammonia,  as  is  shown  in  the  equation  : 

X)H  .OH 

C,;Ht  +  H20  +  O  =  C6H4  +  CH3.COOH  +  NH3. 

eiL.CH(XH,).COOH  XSOOH 

On  oxidation  with  potassium  bichromate  and  sulphuric  acid  hydro- 
cyanic acid,  benzoic  acid,  acetic  acid,  and  formic  acid  result.  - 

With  acids  and  alkalies,  as  also  with  certain  salts  of  the  heavy 
metal-,  tyrosin  combines  with  difficulty  to  form  salt-like  bodies. 

Tests  for  Tyrosin. — Hoffmann's  Test. — With  Millon's  reagent 
tj rosin  gives  the  well-known  reaction  of  those  albumins  in  which 
a.'/omatic  groups  arc  present,  but,  as  would  be  expected,  in  a  degree 
n,uch  more  intense.  The  reaction  is  due  primarily  to  the  formation 
0 .'  oxy-benzoic  acid  (salicylic  acid). 

Pikia's  Test. — A.  i'cw  crystals  of  tyrosin  are  dissolved  in  con- 
centrated sulphuric  acid  and  heated  to  about  100°  C,  when  the  sub- 
stance dissolves.  Tyrosin-sulphuric  acid  is  thus  formed,  and  gives 
rise  to  a  red  color.  ( )n  cooling,  the  liquid  is  diluted,  and  treated  with 
barium  carbonate  while  heating  until  the  reaction  becomes  just 
alkaline.  Tyrosin-sulphate  of  barium  tlms  results,  which  gives  rise 
to  a  dark-violet  color  on  treating  with  a  very  dilute  solution  of 
sesquichloride  of  iron.  An  c\i-c<>  ul'  iron,  however,  must  be  care- 
fully avoided.     Oxy-benzoic  acid  gives  the  same  reaction. 

Scheber's  Test.— On  evaporating  tyrosin  with  a  few  drops  of 

nitric  acid  on  platinum  foil  a  yellow,  transparent  residue  is  obtained, 
which  turn-  ro\  on    moistening  the  substance  with  a  drop  of  sodium 

hydrate  solution,  and  becomes  brown  on  further  evaporation.  The 
reaction  is  due  to  the  formation  of  nitro-tyrosin  nitrate,  but  is  not 
characteristic,  as  other  bodies  behave  in  a  similar  manner. 

Isolation  of  Leucin   and  Tyrosin. — To   isolate   Leucin   and    tyrosin 


190  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

among  the  final  products  of  tryptic  digestion,  and  to  separate  the 
two  from  each  other,  the  digestive  mixture  is  first  freed  from  alkaline 
albuminate,  coagulable  albumins,  and  the  albumoses,  as  already 
described  (page  185).  The  final  filtrate  is  concentrated  to  a  syrupy 
consistence,  when  on  cooling  leucin  and  tyrosin  crystallize  out.  The 
mass  of  crystals  is  then  boiled  with  a  large  quantity  of  water,  to 
which  a  sufficient  amount  of  ammonia  is  added  to  insure  solution  of 
the  substances.  The  boiling  solution  is  treated  with  subacetate  of 
lead  until  the  resulting  precipitate  appears  almost  white.  The  fil- 
trate is  brought  to  the  boiling-point,  neutralized  with  sulphuric  acid, 
and  filtered  while  boiling  hot.  On  cooling,  the  tyrosin  crystallizes 
out,  while  the  leucin  remains  in  solution.  The  former  can  then  be 
purified  by  recrystallization  from  boiling  water  or  from  very  dilute 
ammonia.  The  solution  which  contains  the  leucin  is  freed  from  lead 
with  hydrogen  sulphide,  and  the  filtrate  is  concentrated  and  boiled 
with  an  excess  of  freshly  precipitated  cupric, hydrate.  A  portion  of 
the  leucin  is  thus  precipitated,  while  the  rest  remains  in  solution, 
but  partly  crystallizes  out  on  cooling  as  the  corresponding  copper 
compound.  The  precipitate  is  placed  in  the  copper-containing 
solution,  and  is  freed  from  copper  with  hydrogen  sulphide  ;  the 
filtrate  is  then  decolorized  with  animal  charcoal,  strongly  concen- 
trated, and  set  aside  for  crystallization. 

Asparaginic  Acid. — While  asparaginic  acid  is  apparently  formed 
from  all  albuminous  substances  on  digestion  with  trypsin,  the  largest 
amounts  are  obtained  from  fibrin  and  gelatin.  Like  leucin  and 
tyrosin,  it  likewise  results  on  artificial  decomposition  of  the  albu- 
mins with  dilute  mineral  acids  and  alkalies,  and  is  also  formed 
during  the  process  of  albuminous  putrefaction.  Outside  the  in- 
testinal canal  asparaginic  acid  has  not  been  found  in  the  animal 
body.  In  the  form  of  its  amide  asparagin,  however,  it  is  widely 
distributed  in  the  vegetable  world,  and  supposedly  plays  an  im- 
portant role  in  the  synthesis  of  the  vegetable  albumins. 

The  substance  crystallizes  in  rhombic  prisms,  which  are  soluble 
with  difficulty  in  cold  water,  but  are  quite  soluble  in  hot  water.  In 
absolute  alcohol  it  is  insoluble.  Its  aqueous  solutions  are  lsevorota- 
tory,  while  in  the  presence  of  nitric  acid  dextrorotation  is  observed. 

As  has  been  shown  (page  86),  asparaginic  acid  is  a  dibasic  acid 
of  the  fatty  series.  It  is  amido-succinic  acid,  and  is  represented  by 
the  formula  CH2.CH(NH2).(COOH)2.  It  can  be  obtained  from 
asparagin  on  boiling  with  hydrochloric  acid,  as  shown  in  the 
equation  : 

/CONH2  /COOH 

CH2.CH(NH„)<  +    H20  =  CH2.CH(NH2)<  +   NH3. 

"    \COOH  \COOH 

Asparagin. 

It  has  also  been  produced  synthetically.  On  reduction  it  yields 
succinic  acid,  as  has  been  shown. 

With  cupric  oxide  asparaginic  acid  forms  a  crystalline  compound 


THE  PRODUCTS  OF  TRYPTIC  DIGESTION.  191 

"which  is  almost  insoluble  in  cold  water,  but  dissolves  in  boiling 
"water  with  comparative  ease.  This  property  is  utilized  for  the  pur- 
pose of  isolating  the  substance  from  the  mixture  of  digestive 
products. 

Glutaminic  Acid. — Whether  or  not  glutaminic  acid  is  formed 
during  the  trvptic  digestion  of  the  albumins  in  general  has  not  as 
yet  been  ascertained.  Kutscher  claims  to  have  found  it  in  the 
so-called  antipeptone  of  Kuhne,  which  was  obtained  from  fibrin. 
On  boiling  with  strong  mineral  acids,  however,  it  is  constantly 
formed.  But  it  is  noteworthy  that  much  larger  quantities  are  found 
if  the  decomposition  of  the  albumins  is  effected  with  hydrochloric 
acid  than  with  sulphuric  acid.  Kutscher  thus  found  only  1.8  per 
cent,  among  the  decomposition-products  of  casein  when  using  sul- 
phuric acid,  while  Hlasiwez  and  Habermann  obtained  as  much  as 
29  per  cent,  when  hydrochloric  acid  was  used.  This  is,  of  course, 
remarkable,  and  it  would  be  exceedingly  interesting  to  ascertain  the 
fate  of  those  radicles  which  can  yield  so  large  an  amount  of  gluta- 
minic acid  when  decomposition  is  effected  by  hydrochloric  acid. 

Glutaminic  acid  crystallizes  in  small  glistening  crystals,  whi^h 
are  soluble  with  difficulty  in  cold  water,  while  in  boiling  water  they 
dissolve  with  greater  ease,  but  separate  out  on  cooling.  With  acids 
and  alkalies  it  combines  to  form  salt-like  products,  among  which  t/ie 
hydrochlorate  is  conveniently  utilized  for  the  purpose  of  identifying 
the  substance.     The  melting-point  of  this  compound  is  193°  C. 

The  composition  of  glutaminic  acid  is  expressed  by  the  formula 
CH2.CH2.CH(XH2).(COOH)2.  It  is  thus  amido-glutaric  acid,  and 
bears  the  same  relation  to  glutamin  as  that  which  exists  between 
asparaginic  acid  and  asparagin.     This  is  represented  in  the  equation  : 

/conii2  /cooh 

€h2.ch2.chi\h  +  h20  =  ch2.ch2.ch(nh2)<  +  nt13> 

oooh  x:ooh 

Glutamin. 

On  reduction  it  yields  glutaric  acid. 

Isolation  of  Asparaginic  Acid  and  Glutaminic  Acid. — To  isolate  the 
two  acids  in  question  among  the  products  of  tryptic  digestion,  the 
mixture  must  first  be  freed  from  albumins  and  albumoses,  as  has 
been  described.  The  remaining  solution  is  acidified  with  sulphuric 
acid  and  precipitated  with  phosphotungstic  acid.  The  filtrate  is 
freed  from  sulphuric  acid  and  any  excess  of  the  phosphotungstic 
acid  by  means  of  barium  hydrate.  From  the  resulting  filtrate 
leucin  and  tyrosin  are  then  removed  by  concentration.  The  mother- 
liquor  contains  the  glutaminic  acid  and  asparaginic  acid.  These 
are   now    separated    from    each    other   ill   the    following  manner:    the 

diluted  solution  i-  brought  to  the  boiling-point  and  digested  with 
carbonate  of  copper.  It  IS  filtered  while  still  hot,  and  precipitated 
with  subacetate  of  lead,  care  being  taken  to  avoid  an  excess.  This 
precipitate  i-    decomposed  with    hydrogen    sulphide,  and    the    filtrate 

concentrated  to  a  -mall  volume.     On  standing,  a  crystalline  mass  is 


192  THE  PRODUCTS  OF  ALBUMINOUS  DIGESTION. 

obtained,  which  is  then  dissolved  in  boiling  water  and  digested  with 
an  excess  of  carbonate  of  copper,  as  before.  The  hot  filtrate  is 
again  concentrated,  when  on  standing  the  copper  salt  of  asparaginic 
acid  separates  out  in  characteristic  groups  of  needles.  The  nitrate 
is  freed  from  copper  by  means  of  hydrogen  sulphide,  concentrated,, 
and  set  aside,  when  the  glutaminic  acid  crystallizes  out. 

Glycocoll. — While  it  is  generally  known  that  glycocoll  plays  an 
important  part  in  the  nitrogenous  metabolism  of  the  animal  body, 
and  is  intimately  concerned  in  the  formation  of  urea,  hippuric  acid, 
phenaceturic  acid,  certain  biliary  acids,  and  in  birds  and  reptiles  of 
uric  acid,  it  is  of  interest  to  note  that  the  substance  has  thus  far 
not  been  found  as  such  among  the  products  of  pancreatic  digestion, 
although  its  radicle  is  manifestly  present  in  certain  albumoses.  On 
hydrolytic  decomposition  with  mineral  acids,  on  the  other  hand, 
glycocoll  can  be  obtained  from  most  albumins,  but  is  especially 
abundant  in  collagen,  viz.,  gelatin.  Two  exceptions  to  this  general 
rule,  however,  are  noted,  viz.,  casein  and  (according  to  Magnus- 
Levy)  the  peculiar  albuminous  substance  which  is  known  as  the 
Bence  Jones'  body,  and  from  either  of  these  it  is  also  impossible  to 
obtain  a  hetero-albumose.  The  hetero-albumose  of  fibrin,  according 
to  Spiro,  yields  a  considerable  amount  of  glycocoll,  while  from  the 
proto-albumose  it  cannot  be  obtained. 

Heretofore  the  isolation  of  glycocoll  and  its  recognition  as  such, 
were  attended  with  great  difficulties.  A  somewhat  simpler  pro- 
cedure, however,  has  recently  been  suggested  by  Baum,  and  with 
its  aid  Spiro  was  able  to  show  that,  contrary  to  former  views,  the 
substance  can  be  obtained  not  only  from  the  albuminoids,  but  also 
from  the  native  albumins,  with  the  exceptions  indicated.  t  The 
method  is  based  upon  the  observation  that  glycocoll  can  be  trans- 
formed into  hippuric  acid  in  the  test-tube  by  treating  with  benzoyl 
chloride  in  the  presence  of  sodium  hydrate,  and  that  the  formation 
of  the  resulting  hippuric  acid  can  be  readily  demonstrated  by  con- 
densing this  with  benzaldehyde  in  the  presence  of  sodium  acetate 
and  acetic  anhydride.  The  lactimide  of  benzoyl-amido-cinnamic 
acicl  is  thus  formed.  On  decomposition  with  sodium  hydrate  this 
yields  phenyl-pyro-racemic  acid,  which  in  ethereal  solution  gives  a. 
green  color  on  treating  with  chloride  of  iron.  With  phenyl- 
bydrazin,  moreover,  it  forms  an  osazon  which  melts  at  161°  C. 
These  changes  may  be  represented  by  the  equations  : 

(1)  CH2.(NH2).COOH  +  C6H5.C0C1  =  CH2.NH(C6H5.CO).COOH  +  HC1 

Glycocoll.  Benzoyl  chloride.  Hippuric  acid. 

(2)  CH2.NHfC,H5.CO).COOH  4-  C6H5COH  =  C6H5.CO.N.C:CH.C6H5  +  2H20 

Hippuric  acid.  Benzaldehyde.  |  / 

CO 

Lactimide. 

(3)  C6H5.CO.N.C:CH.C6H5  +  H20  =  C6H5.CO.NH2.C.C6H5.CH.COOH 

\/  Benzoyl-amido-cinnamic 

p,,-.  acid. 

Lactimide. 


THE  PRODUCTS   OF  TRYPTIC  DIGESTION.  193 

(4)  C6H3CO.XH2.C.C6H5.CH.COOH  +  H20  = 

Benzoyl-amido-ciunamic  acid.  <J6H5.CO.XH2  +  C6H5.CH,.CO.COOH 

Benzainide.  Phenyl-pyro-racemic 

acid. 

Method. — The  decomposition  of  the  albumins  (gelatin)  is  effected 
bv  prolonged  boiling  with  dilute  sulphuric  acid — 25  percent,  solution. 
The  excess  of  acid  is  removed  with  plumbic  carbonate.  The  nitrate 
is  concentrated,  freed  from  any  tyrosin  that  may  have  separated  out, 
and  then  benzoylated  with  benzoyl  chloride  in  the  presence  of 
sodium  hydrate.  Care  should  be  had  that  the  reaction  of  the  solu- 
tion is  constantly  alkaline  during  this  process.  The  hippuric  acid 
is  then  extracted  with  acetic  ether.  The  dried  substance  is  now 
treated  with  three  molecules  of  acetic  anhydride,  one  molecule  of 
sodium  acetate,  and  one  molecule  of  benzaldehyde.  The  mixture  is 
heated  on  a  water-bath  for  half  an  hour.  The  condensation-produce 
is  then  treated  with  water  and  gently  warmed.  The  oil  that  sepa- 
rates out  is  dissolved  in  hot  alcohol  and  allowed  to  cool.  The  lacti- 
mide  then  crystallizes  out,  and  can  be  recognized  as  follows :  the 
substance  is  heated  with  a  strong  solution  of  sodium  hydrate 
until  a  distinct  odor  of  ammonia  is  noticed.  This  is  due  to 
the  decomposition  of  the  benzamide.  On  acidifying  the  solution 
the  phenyl-pyro-racemic  acid  separates  out  and  can  be  readily  ex- 
tracted by  shaking  with  ether.  One  portion  of  the  ethereal  extract 
is  treated  with  a  dilute  solution  of  the  sesquichloride  of  iron,  when 
on  agitation  the  watery  layer  assumes  a  dark-green  color,  which 
gradually  changes  to  a  characteristic  yellow.  The  other  portion  is 
treated  with  an  ethereal  solution  of  phenylhydrazin,  which  leads  to  the 
separation  of  the  hydrazon  of  phenyl-pyro-racemic  acid.  After  wash- 
ing with  ether  this  may  be  identified  by  its  melting-point — 161°  C. 

As  regards  the  general  properties  of  glycocoll  and  its  preparation 
as  such,  sec  pages  87  and  2o9). 

Tryptophan. — This  substance  is  apparently  always  formed  when 
the  tryptie  digestion  of  the  albumins  has  extended  beyond  the  forma- 
tion of  albumoses.  As  its  presence  among  the  various  digestive 
products  is  easily  recognized,  it  is  thus  possible  to  ascertain  whether 
the  destruction  of  the  albuminous  molecule  has  extended  to  the 
formation  of  amido-acids,  without  testing  for  these  directly.  Like 
the  amido-acids,  if  is  also  formed  during  the  hydrolytic  decomposi- 
tion of  the  albumins  with  baryta-water,  and  likewise  results  during 
the  process  of  intestinal  putrefaction.  Of  special  interest  is  the  fact 
that  while  the  primary  albumoses  of  fibrin,  as  also  the  secondary 
albumose-A,  on  further  digestion  with  trypsin,  give  rise  to  the 
formation  of  tryptophan,  the  secondary  albumose-B'  at  least  appar- 
ently does  not  contain  the  chromogenic  group. 

The  substance  itself  ie  colorless,  and  is  hence  also  spoken  of  as 
proteinochromogen.  With  chlorine  and  bromine  it  yields  at  least 
three  colored  products,  the  proteinochromes,  and  it  is  hence  supposed 
thai    several   varieties  of  the   chromogen   may  also  exist,     or  the 

chemical    nature    of    both,    however,    but     little    is    known.      While 


194  THE  PRODUCTS   OF  ALBUMINOUS  DIGESTION. 

according  to  some  observers  the  entire  amount  of  sulphur  is  split 
off  from  the  albuminous  molecule  in  the  form  of  the  chromogen, 
others  maintain  that  the  sulphur  of  the  proteinochromes  is  referable 
to  contamination  with  other  substances. 

With  bromine  three  pigments  at  least  may  be  obtained,  viz.,  a 
bluish-violet  substance,  which  contains  about  35  per  cent,  of  bro- 
mine ;  a  red  body,  with  27  per  cent.;  and  a  brown  pigment,  with  the 
same  amount  of  bromine.  The  violet  pigment,  moreover,  is  said  to 
contain  a  considerable  amount  of  iron,  but  it  is  noteworthy  that 
albumins  which  are  free  from  iron  also  give  rise  to  the  formation 
of  proteinochromes. 

Breitler  has  isolated  a  chloroproteinochrome,  to  which  he  gives 
the  formula  C96HU9N2103S.  This  does  not  coincide  with  any  one  of 
the  three  bodies  that  have  been  just  referred  to,  but  it  is  quite  pos- 
sible that  still  other  chromogens  exist. 

According  to  JN"encki,  a  certain  similarity  exists  in  the  percentage 
-composition  of  the  red  pigment  with  hsemoporphyrin,  viz.,  bilirubin, 
and  of  the  brown  pigment  with  the  so-called  melanins.  The  tryp- 
tophan, moreover,  like  hsematin  and  hseinatoporphyrin,  yields  pyrrol, 
hydrogen  sulphide,  methyl-mercaptan,  and  skatol  on  fusing  with 
caustic  alkali. 

Test. — The  test  for  tryptophan  and  the  isolation  of  the  three 
known  pigments  are  conducted  as  follows :  the  digestive  mixture 
is  acidified  with  acetic  acid  and  treated  with  two  and  one-half  times 
its  volume  of  saturated  bromine-water.  A  beautiful  reddish-violet 
precipitate  is  thus  formed,  which  increases  on  standing.  After 
twenty-four  hours  this  is  filtered  off.  On  the  further  addition  of 
bromine-water  the  brown  pigment  separates  out  on  standing.  The 
red  pigment  will  be  found  in  the  violet  precipitate,  and  can  be  iso- 
lated as  follows  :  the  precipitate  is  first  washed  with  water  and  then 
extracted  with  dilute  ammonia ;  this  extract  is  precipitated  with 
acetic  acid.  The  precipitate  is  separated  from  the  brown  filtrate, 
redissolved  in  very  dilute  ammonia,  again  precipitated  with  acetic 
acid,  and  washed  with  water.  It  is  then  extracted  with  amyl 
alcohol ;  this  dissolves  the  red  body.  The  alcohol  is  evaporated 
off  at  40°  C,  the  residue  dried  at  106°  C,  and  finally  washed  with 
ether.  The  violet  pigment  is  obtained  on  further  extraction  of  the 
violet  precipitate  with  a  little  stronger  solution  of  ammonia  than  in 
the  first  instance.  The  substance  is  precipitated  with  acetic  acid, 
well  washed  with  water,  and  extracted  with  95  per  cent,  alcohol. 
The  alcoholic  extract  is  evaporated  to  dryness  at  40°  C,  the  residue 
dried  at  106°  C.  and  washed  with  petroleum  ether. 

To  isolate  the  brown  pigment,  finally,  the  second  bromine  pre- 
cipitate is  filtered  off,  washed  with  water,  dissolved  in  very  dilute 
ammonia,  reprecipitated  with  acetic  acid  and  washed  with  water, 
and  briefly  with  95  per  cent,  alcohol,  both  of  which  dissolve  a  por- 
tion of  the  pigment.  It  is  then  dried  and  washed  with  ether.  The 
resulting  product  is  almost  black. 


THE  PRODUCTS  OF  TBYPTIC  DIGESTION.  195 

Antipeptone. — To  prepare  antipeptone  in  amounts  which  are 
sufficient  for  the  purpose  of  isolating  the  hexon  bases  which  the  sub- 
stance supposedly  contains,  it  is  necessary  to  start  with  a  large  quantity 
of  fibrin  :  1000  grammes  of  the  latter  are  suspended  in  2000  c.c.  of 
an  0.25  per  cent,  solution  of  sodium  carbonate,  to  which  a  few 
grammes  of  an  active  pancreatin  preparation  have  been  added. 
Putrefaction  is  prevented  by  adding  an  amount  of  chloroform  suffi- 
cient to  saturate  the  solution,  as  also  a  few  crystals  of  thymol.  The 
mixture  is  thoroughly  shaken  and  kept  at  a  temperature  of  40°  C. 
for  at  least  one  week.  It  is  then  filtered,  slightly  acidified  with 
acetic  acid,  boiled,  again  filtered,  and  concentrated  to  about  1000  c.c. 
On  cooling,  a  good  deal  of  tyrosin  separates  out  and  is  filtered  off. 
The  filtrate  is  diluted  with  water  to  about  2000  c.c,  neutralized, 
heated  to  near  the  boiling-point,  and  saturated  with  ammonium  sul- 
phate in  substance.  On  cooling,  any  albumoses  that  may  have  sepa- 
rated, together  with  a  large  quantity  of  the  salt,  are  filtered  off.  The 
filtrate  is  heated,  and  while  boiling  rendered  strongly  alkaline  with 
ammonia  and  ammonium  carbonate,  and  again  saturated  with  ammo- 
nium sulphate.  On  cooling,  a  second  fraction  of  albumoses  is  filtered 
off.  The  solution  is  then  heated  until  the  odor  of  ammonia  has  dis- 
appeared ;  ammonium  sulphate  is  again  added  to  saturation,  and  the 
liquid  rendered  distinctly  acid  with  acetic  acid,  when  on  cooling  a 
third  fraction  of  albumoses  separates  out  and  is  filtered  off.  The 
filtrate  is  concentrated  to  about  one  liter  and  freed  from  a  large 
amount  of  ammonium  sulphate,  which  separates  out  on  cooling.  It 
is  then  diluted  with  water  to  about  3000  c.c,  and  treated  at  a  tem- 
perature of  30°  C,  with  barium  hydrate  in  substance,  to  remove 
the  remaining  salt.  A  slight  excess  of  the  barium  is  removed  with 
carbonic  acid,  and  is  filtered  off  after  boiling  for  a  moment.  The 
filtrate  is  evaporated  to  about  1000  c.c,  when  the  barium  peptone  is 
decomposed  with  dilute  sulphuric  acid,  care  being  taken  that  the 
acid  is  not  added  in  excess.  The  resulting  barium  sulphate 
is  filtered  off  and  the  filtrate  concentrated  to  a  thin  syrup.  On  cool- 
ing, absolute  alcohol  is  added  until  the  turbidity  that  iirst  appears 
do  longer  disappears  on  stirring.  After  filtering  with  the  aid  of  a 
suction  |)iini]>,  the  solution  is  poured  into  absolute  alcohol  while 
stirring.  The  antipeptone  is  then  precipitated  and  allowed  to  settle, 
when  the  supernatant  fluid  is  siphoned  off  and  the  antipeptone  col- 
lected on  a  filter  with  the  aid  of  a  suction-pump.  It  is  finally 
washed  with  absolute  alcohol,  then  with  ether,  and  rapidly  placed  in 
a  desiccator  over  sulphuric  acid. 

From  this  material  Kutscher  claims  that  the  three  hexon  bases 
can  then  be  isolated.  To  demonstrate  that  these  bodies  actually 
result  from  the  albuminson  hydrolytic decomposition,  it  is  more  con- 
venient, however,  to  effect  this  by  boiling  with  dilute  acids.  To 
this  end,  commercial  gelatin   is  conveniently  utilized  as  a  starting 

material,  a-  larger  amounts  of*  arginin  at   least  can  thus  be  obtained, 

The  method,   however,  is  quite  complicated  and  scarcely  requires 
consideration  at  this  place. 


CHAPTEK    X. 

BACTERIAL  ACTION  IN  THE  INTESTINAL  TRACT. 

I  have  pointed  out  in  a  preceding  chapter  that  the  gastric  juice 
possesses  marked  germicidal  and  antiseptic  properties,  so  that  a  large 
number  of  bacteria  which  are  constantly  swallowed  with  the  saliva 
and  the  food  are  subsequently  destroyed  in  the  stomach.  A  perfect 
barrier  to  the  invasion  of  micro-organisms,  however,  does  not  exist, 
and  after  having  passed  the  pylorus  they  are  placed  in  surround- 
ings which  are  in  all  respects  most  favorable  to  their  develop- 
ment. Here  they  take  an  active  part  in  the  decomposition  of  the 
various  food-stuffs  which  have  escaped  digestion  in  the  stomach,  and 
further  modify  the  digestive  products  which  have  already  been 
formed,  as  also  those  which  result  from  the  action  of  the  various 
intestinal  ferments.  The  greater  portion  of  the  products  of  normal 
digestion,  however,  escapes  the  specific  activity  of  the  bacteria,  and 
is  absorbed  in  a  form  which  can  be  utilized  by  the  body  for  purposes 
of  nutrition.  Formerly  it  was  supposed  that  the  biliary  acids  played 
an  important  part  in  preventing  undue  activity  on  the  part  of  the 
bacteria,  but  this  view  has  now  been  largely  abandoned,  and  we  are 
totally  ignorant  as  to  the  manner  in  which  the  body  here  protects 
itself  against  excessive  bacterial  action.  It  has  been  argued  that  an 
accumulation  of  the  decomposition-products  which  result  from  the 
action  of  bacteria  upon  the  various  food-stuffs  in  itself  inhibits  the 
further  activity  of  the  organisms,  but  we  can  hardly  regard  such  an 
explanation  as  valid  in  view  of  the  fact  that  in  the  intestines  these 
decomposition-products  are  to  a  large  extent  absorbed,  and  it  seems 
more  probable  that  a  vital  activity  of  the  epithelial  cells  is  here 
of  prime  importance.  In  the  small  intestine  at  least,  where  peri- 
stalsis is  extremely  active,  and  where  the  intestinal  contents  are 
churned  in  such  a  manner  that  the  individual  particles  are  almost 
constantly  in  contact  with  the  intestinal  walls,  we  accordingly  find 
that  bacterial  action  is  not  nearly  so  extensive  as  in  the  large  intes- 
tine, where  the  opposite  conditions  prevail.  In  the  clinical  labo- 
ratory we  find,  as  a  matter  of  fact,  that  the  degree  of  intestinal 
putrefaction  increases  at  once  when  the  peristalsis  of  the  small 
intestine  is  impeded,  and  reaches  its  greatest  height  if  the  secretion 
of  hydrochloric  acid  becomes  arrested  at  the  same  time. 

In  former  years  a  tendency  existed  among  physiologists  to  regard 
bacterial  action  in  the  intestine  as  serving  a  useful  purpose,  and  it 
was  even  supposed  that,  as  in  the  case  of  plants,  animal  life  could 
not  go  on  in   the  absence  of  micro-organisms  from  the  alimentary 

196 


BACTERIAL   ACTIOS  IN   THE  INTESTINAL   TRACT.         197 

canal.  This  view  has  now  been  abandoned,  however,  especially 
since  Thierfelder  and  Xuttall  were  able  to  demonstrate  that  guinea- 
pigs,  after  removal  from  the  uterus  of  the  mother  by  Csesarean 
section,  can  be  maintained  in  perfect  condition  as  to  health  and 
body-weight  when  fed  on  sterile  food  and  when  furnished  with 
sterile  air  exclusively.  On  subsequent  examination  it  was  shown 
that  the  intestinal  contents  of  these  animals  were  also  sterile.  We 
may  thus  conclude  that  the  presence  of  bacteria  in  the  intestinal 
contents  is  at  best  unnecessary,  and  it  is  doubtful,  indeed,  whether 
they  serve  a  useful  purpose  at  any  time. 

The  action  of  bacteria  upon  the  food-stuffs  is  in  certain  respects 
quite  analogous  to  that  of  the  digestive  ferments  which  are  fur- 
nished by  the  digestive  glands  of  the  animal  body.  The  primary 
digestion  of  the  original  material,  however,  does  not  cease  with  the 
production  of  substances  which  the  animal  can  subsequently  utilize 
for  the  purpose  of  replacing  tissue,  but  is,  on  the  whole,  far  more 
extensive.  Polysaccharides  and  disaccharides  are  thus  not  only 
inverted  to  monosaccharides,  but  the  latter  are  subsequently  further 
decomposed  into  material  in  which  but  little  potential  energy,  if 
any,  remains  stored.  Albumins  are  similarly  decomposed,  with 
the  ultimate  formation  of  substances  which  in  part  at  least  are  dis- 
tinctly toxic  ;  and  the  fats  are  divided  into  their  components,  which 
are  then  further  broken  down,  with  the  final  formation  of  fattv 
acids  of  the  lowest  order,  etc.  A  great  variety  of  decomposition- 
products  thus  result  from  the  normal  food-stuffs,  which  are  further 
increased  by  those  arising  from  material  which  the  ferments  of 
the  animal  itself  are  incapable  of  digesting.  To  these  are  added 
the  decomposition-products  of  the  various  biliary  constituents  and 
of  the  albuminous  secretions  which  are  poured  into  the  intestinal 
canal  by  the  digestive  glands  themselves. 

A-  has  been  pointed  out,  the  most  intense  degree  of  bacterial 
action  is  observed  in  the  large  intestine,  and  it  is  interesting  to 
note  that  while  albuminous  putrefaction  here  prevails,  the  fermenta- 
tive processes  in  the  more  restricted  sense  of  the  term,  viz.,  the 
decom]K>sition  of  carbohydrates  and  fats,  occur  almost  exclusively  in 
the  small  intestine.  This  difference  may  be  dependent  to  a  certain 
degree  upon  the  difference  in  the  reaction  of  the  intestinal  contents 
in  the  two  sections  of  the  gut — that  of  the  small  intestine  in  its  lower 
portion  at  least  being  acid,  while  the  reaction  of  the  contents  of  the 
large  intestine  is  usually  alkaline.  But  it  is  also  possible  that  other 
and  -till  unknown  factors  determine  this  difference,  ami  that  the 
varying  reaction  is  primarily  due  to  the  decomposition-products 
directly  which  result  from  the  action  of  the  bacteria.  Among  these 
factors  tli''  relative  amount  of  water  may  be  of  importance. 

Nencki,  MacFadyen,  and  Sieber,  who  had  occasion  to  study  the 
chemical  composition  of  the  intestinal  contents  in  ;i  patient  in  whom 
an  artificial  ami-  had  been  established  at  the  distal  end  of  the  ileum, 
give  the  following  account  of  their  observations  :     The  reaction  was 


198        BACTERIAL  ACTION  IN  THE  INTESTINAL   TRACT. 

quite  constantly  acid,  owing  to  the  presence  of  organic  acids,  and 
notably  of  acetic  acid.  Other  acids  that  were  present  were  lactic 
acid,  paralactic  acid,  various  volatile  fatty  acids,  succinic  acid,  and 
the  biliary  acids.  The  odor  but  rarely  suggested  the  existence  of 
putrefactive  changes.  Indol,  skatol,  and  phenol  could  not  be 
demonstrated  as  such,  although  the  urine  contained  indican  on 
several  occasions.  Leucin  and  tyrosin  were  not  found.  Alcohol 
could  always  be  demonstrated.  Of  gases,  carbon  dioxide  was  ob- 
served, as  also  faint  traces  of  hydrogen  sulphide,  while  methyl- 
mercaptan  was  absent. 

Carbohydrate  fermentation  thus  manifestly  stands  in  the  fore- 
ground, and  is  exemplified  in  various  types  by  the  equations : 

(1)  C6H1206  =  2C2H5.OH  +  2C02,  alcoholic  fermentation. 

(2)  C2H5.OH  +  20  =  CHg.COOH  +  H20,   acetic  acid  fermentation. 

(3)  C6HI206  =  2CH3.CH2(OH)COOH,  lactic  acid  fermentation. 

(4)  2C3H603  =  C3H7.COOH  +  2C02  +  4H,  butyric  acid  fermentation. 

The  products  of  albuminous  putrefaction,  on  the  other  hand,  are 
almost  exclusively  formed  in  the  large  intestine.  Primarily  they 
are  in  part  at  least  the  same  as  those  which  result  from  the  action 
of  trypsin  on  albumins,  and  in  experiments  in  vitro  we  thus  find 
albumoses,  peptone-like  bodies,  tryptophan,  leucin,  tyrosin,  aspara- 
ginic acid,  and  glutaminic  acid.  In  the  contents  of  the  large  intes- 
tine, however,  these  substances  are  found  only  in  traces,  so  that  we 
are  forced  to  the  conclusion  that  they  are  either  absorbed  as  soon  as 
formed  or  that  they  are  further  decomposed.  Both,  no  doubt, 
occurs,  and  related  bodies  are,  as  a  matter  of  fact,  encountered  in 
the  feces.  As  a  result  of  bacterial  activity  still  other  substances 
are  formed,  however,  which  are  apparently  not  derived  from  the 
final  products  of  digestion,  but  which  are  formed  from  the  more  or 
less  intact  albuminous  molecule  directly. 

The  more  important  decomposition-products  which  result  from 
the  action  of  bacteria  upon  the  products  of  albuminous  digestion  are 
here  considered. 

Indol. — We  have  seen  that  on  decomposition  of  the  albumins 
with  trypsin,  as  also  with  boiling  mineral  acids,  the  aromatic 
groups  of  the  albuminous  molecule  are  split  off  in  the  form  of 
tyrosin — i.  e.,  a  body  belonging  to  the  para-series.  Indol,  on  the 
other  hand,  belongs  to  the  ortho-series,  and  cannot  be  obtained  in 
this  manner.  It  is  a  specific  product  of  albuminous  putrefaction, 
and  it  would,  of  course,  be  interesting  to  ascertain  why  the  aromatic 
groups  in  the  one  case  are  set  free  exclusively  in  the  form  of 
tyrosin,  while  in  the  other  both  result  side  by  side.  At  present  we 
are  unable  to  offer  an  adequate  explanation  of  this  phenomenon,  but 
it  is  possible,  as  Neumeister  suggests,  that  certain  bacteria  produce 
indol  synthetically  from  simpler  aromatic  groups.     We  find,  as  a 


SKATOL.  199 

matter  of  fact,  that  under  the  influence  of  certain  organisms,  such 
as  the  Proteus  vulgaris,  indol  is  formed  almost  exclusively. 

Structurally  indol  is  closely  related  to  indigo,  and  according  to 
Xeneki  this  transformation  can  be  effected  through  the  action  of 
ozone.     It  is  represented  by  the  equation  : 

2C6H4<         >CH    +   40   =   C6H  /         >C  =  C<         }C,H4  +   2H20. 
XII  XNH7  XNHX 

Indol.  Iudigo. 

(  inversely,  indigo  can  be  transformed  into  indol  on  reduction. 

From  the  albumins  the  substance  can  also  be  obtained  on  fusion 
with  potassium  hydroxide  (see  page  38). 

The  greater  portion  of  the  indol  that  is  formed  in  the  large  in- 
testine is  no  doubt  eliminated  in  the  feces.  A  certain  amount, 
however,  is  absorbed,  and  after  oxidation  to  indoxyl  appears  in  the 
urine  in  combination  with  sulphuric  acid  as  so-called  indican 
(pages  90  and  250).  If  larger  quantities  are  formed,  a  variable 
fraction  is  further  eliminated  in  the  urine  as  an  indoxyl  compound 
of  glucuronic  acid. 

Indol  crystallizes  in  small  platelets,  which  melt  at  52°  C,  and 
are  soluble  in  hot  water,  ether,  alcohol,  and  benzol.  Its  odor  is 
feculent ;  it  is  quite  volatile,  and  when  boiled  with  water  passes  over 
into  the  distillate.  With  picric  acid  it  forms  a  beautifully  red  crys- 
talline compound,  which  is  readily  decomposed,  however,  on  boil- 
ing  with  dilute  ammonia,  and  the  liberated  indol  is  then  found  in 
the  distillate.  On  distilling  in  the  presence  of  sodium  hydrate,  on 
the  other  hand,  the  indol  is  decomposed. 

Tests. — When  treated  in  aqueous  solution  with  nitric  acid  and  a 
trace  of  sodium  nitrite  a  red  precipitate  of  the  nitrate  of  nitroso- 
indol  is  formed.  This  is  soluble  in  alcohol  and  crystallizes  out  upon 
the  addition  of  ether. 

If  a  small  piece  of  pine  wood  is  moistened  with  strong  hydro- 
chloric acid  and  then  placed  in  a  watery  solution  of  indol,  it  gradu- 
ally assumes  a  cherry-red  color. 

An  aqueous  solution  of  indol  is  treated  with  a  small  amount  of 
a  solution  of  sodium  nitroprusside  until  a  brownish-yellow  color 
develops.  If  now  a  dilute  solution  of  sodium  hydrate  is  added 
drop  by  drop,  the  color  changes  to  violet.  Upon  the  further  addi- 
tion of  a  little  dilute  hydrochloric  acid  this  becomes  a  deep  blue, 
while  an  exce88  of  the  ;teid  destroys  the  blue  color. 

Por  the  isolation  of  indol,  see  page  206). 

Skatol. — Skatol,  like  indol,  belongs  to  the  ortho-series,  and  is 
likewise  formed  during  the  process  of  albuminous  putrefaction.  It 
i-  a  methylated  indol,  and  may  !"■  represented  by  the  formula: 


C(CH 

MI 

By  combining  with  carbon  dioxide  it  gives  rise  to  the  formation 


"MM 

'•,.11,  CH. 

MI 


200        BACTERIAL  ACTION  IN  THE  INTESTINAL   TRACT. 

of  skatol-carbonic  acid,  which   is  also  found  in  the  contents  of  the 
large  intestine,  and  belongs  to  the  ortho-series.     Its  formula  is 

/C(CH3U 
C6H4<  ^C.COOH. 

Like  indol,  skatol  is  also  formed  on  fusing  albumins  with  caustic 
soda,  and  can  be  obtained  from  indigo  on  reduction  with  tin  and 
hydrochloric  acid.  When  passed  through  a  red-hot  tube  it  yields 
indol.  On  absorption,  it  is  oxidized  to  skatoxyl  and  is  eliminated  in 
the  urine  in  combination  with  sulphuric  acid  and  glucuronic  acid,  as 
in  the  case  of  indol  (see  pages  90  and  250).  Skatol-carbonic  acid,  on 
the  other  hand,  appears  in  the  urine  as  such. 

Skatol  crystallizes  in  fine  platelets,  which  melt  at  95°  C.  and  are 
readily  soluble  in  ether,  alcohol,  and  benzol ;  in  hot  water  it  is 
soluble  with  greater  difficulty  than  indol.  Its  odor  is  exceedingly 
offensive.  Like  indol,  it  is  volatile,  and  combines  with  picric  acid 
to  form  a  red  crystalline  compound.  On  distilling  this  in  ammo- 
niacal  solution  or  in  the  presence  of  sodium  hydrate  the  skatol  passes 
over  as  such,  while  indol  in  the  latter  instance  is  decomposed.  On 
distilling  a  mixture  of  indol  and  skatol  in  aqueous  solution  the 
skatol  passes  over  first,  and  it  is  thus  possible  to  separate  the  two 
substances  from  each  other. 

Tests. — From  its  aqueous  solutions  skatol  is  precipitated  by  yellow 
nitric  acid  as  a  white  substance — skatol  nitrate. 

If  a  small  piece  of  pine  wood  is  moistened  with  an  alcoholic  solu- 
tion of  skatol  and  then  placed  in  strong  hydrochloric  acid,  it  assumes 
a  red  color.  If,  on  the  other  hand,  the  test  is  conducted  as  with 
indol,  no  reaction  is  obtained. 

With  nitric  acid  of  a  specific  gravity  of  1.2  skatol  gives  a  marked 
xanthoproteic  reaction  on  boiling — i.e.,  a  yellow  color  which  changes 
to  orange  when  ammonia  is  added  in  excess. 

The  substance  does  not  give  the  reaction  with  sodium  nitro- 
prusside. 

Isolation  (see  page  206). 

Phenol. — The  phenol  which  is  formed  during  the  process  of 
intestinal  putrefaction  is  derived  from  tyrosin.  As  has  been  shown, 
this  is  first  reduced  to  hydroparacumaric  acid.  This  in  turn  is 
oxidized  to  para-oxy-phenyl-acetic  acid.  Paracresol  then  is  formed 
through  a  splitting  off  of  carbon  dioxide,  and  on  subsequent  oxida- 
tion phenol  results  (see  page  96).  To  a  certain  extent  this  is  elimi- 
nated in  the  feces,  but  a  variable  amount  is  always  absorbed,  and 
subsequently  oxidized  in  part  to  hydroquinon  or  pyrocatechin. 
These  three'  bodies  then  combine  with  sulphuric  acid  and  are  elimi- 
nated through  the  urine  in  this  form.  A  certain  amount  of  para- 
cresol, moreover,  is  absorbed  as  such,  and  likewise  appears  in  the 
urine  as  a  conjugate  sulphate.  According  to  some  observers,  indeed, 
a  larger  quantity  of  paracresol  is  here  encountered  than  of  phenol. 


PHENOL.  201 

Tests. — An  aqueous  solution  of  phenol  when  treated  with  a  few 
drops  of  a  solution  of  the  sesquichloride  of  iron  assumes  an  amethyst 
color,  which  becomes  especially  apparent  on  further  dilution  with 
water  if  much  phenol  is  present. 

With  bromine-water  a  crystalline  precipitate  of  tribromophenol  is 
obtained. 

With  Millon's  reagent  a  red  color  develops,  which,  however,  is 
common  to  other  bodies  of  this  series  as  well  (see  page  34). 

Isolation  (see  page  206). 

In  addition  to  phenol,  indol,  skatol,  and  skatol-carbonic  acid,  as 
also  the  two  hvdroxylated  benzol-derivatives  of  tyrosin,  viz.,  para- 
oxy-phenyl-propionic  acid  (hydroparacumaric  acid)  and  para-oxy- 
phenyl-acetic  acid,  we  further  meet  with  two  non-hydroxylated 
aromatic  acids,  which  are  homologous  with  benzoic  acid,  viz.,  phenyl- 
propionic  or  hydrocinnamie  acid  and  phenyl-acetic  acid.  Accord- 
ing to  Salkowski,  these  may  develop  directly  from  the  albuminous 
molecule,  but  may  also  result  from  tyrosin  (see  page  97). 

The  non-nitrogenous  aromatic  acids  are  in  part  eliminated  in  the 
feces.  To  some  extent,  however,  they  are  also  absorbed.  The 
hvdroxylated  acids  are  then  eliminated  in  the  urine  either  as  such, 
or,  like  phenol,  indol,  and  skatoxyl,  in  combination  with  sulphuric 
acid,  while  the  non-hydroxylated  acids  combine  with  glycocoll,  and 
are  eliminated  as  hippuric  acid  and  phenaceturic  acid,  as  already 
described  (page  97). 

As  regards  the  fate  of  the  small  amounts  of  leucin,  asparaginic 
acid,  and  glutaminic  acid  which  are  also  formed  during  the  process 
of  albuminous  putrefaction,  it  seems  that  they  are  usually  absorbed, 
and  are  then  further  decomposed  within  the  body  of  the  animal.  To 
a  slight  extent,  however,  this  decomposition  also  takes  place  within 
the  large  intestine,  and  we  accordingly  meet  with  small  amounts 
of  -uccinic  acid,  glutaric  acid,  eapronic  acid,  valerianic  acid,  butyric 
acid,  and  acetic  acid.  The  sulphur  of  the  albuminous  molecule  is 
usually  set  free  in  the  form  of  hydrogen  sulphide,  but  traces  of 
methyl-mereaptan  are  also  frequently  observed,  and  still  further 
contribute  to  the  offensive  odor  of  the  feces.  Whether  these  sulphur 
bodies  result  from  decomposition  of  the  tryptophan,  is  not  known. 

Of  the  gases  which  are  constantly  present  in  the  contents  of  the 
large  intestine,  methane  further  deserves  especial  mention.  It  is  to 
a  great  extent,  in,  doubt,  referable  to  the  peculiar  form  of  fermenta- 
tion to  which  the  celluloses  are  subject.  But  in  part  at  least  it  prob- 
ably also  results    from    the   decomposition  of  the    fattv  acids  and  of 

cholin. 

Ptomains  are  normally  not  found  in  the  intestinal  contents,  but 
may  l»e  encountered  under  certain  pathological  conditions.  In  Asiatic 
cholera  and  in  cases  ofcystinuria  putrescin  and  cadaverin  have  thus 
been  isolated,  and  in  other  diseases,  no  doubt,  they  also  occur. 

Th"  methods  which  are  employed  fir  the  purpose  of  isolating  the 


202        BACTERIAL  ACTION  IN  THE  INTESTINAL   TRACT. 

more  important  products  of  albuminous  putrefaction  are  described  in 
the  chapter  on  the  Feces. 

BACTERIAL  DECOMPOSITION  OF  THE  FATS. 

As  in  the  case  of  the  carbohydrates  and  albumins,  a  comparatively 
small  portion  of  the  fats  only  undergoes  bacterial  decomposition,  and 
it  appears  that  this  principally  occurs  in  the  lower  portion  of  the 
small  intestine.  As  in  the  case  of  the  steapsin  of  the  pancreatic 
juice,  the  neutral  fats  are  thus  first  decomposed  into  glycerin  aud 
the  corresponding  fatty  acids,  but  the  process  extends  further, 
and  as  a  result  a  gradual  reduction  of  the  higher  acids  to  the  lowest 
forms  takes  place.  To  a  certain  extent  these  are  then  absorbed  and 
further  decomposed  in  the  body,  but  a  not  inconsiderable  portion  is 
directly  eliminated  in  the  feces,  and  we  accordingly  find  here  repre- 
sentatives of  the  group,  from  palmitic,  stearic,  and  oleic  acids  down 
to  butyric  acid  and  acetic  acid.  The  glycerin  is  absorbed,  and  is  to 
a  certain  extent  no  doubt  utilized  by  the  epithelial  cells  in  the  syn- 
thesis of  fats. 

The  lecithins  are  decomposed  in  the  same  manner  as  under  the 
influence  of  steapsin,  with  the  formation  of  glycerin-phosphoric  acid, 
fatty  acids,  and  cholin.  Whether  or  not  the  latter  may  then  be 
transformed  into  neurin  is  not  known,  but  under  normal  condi- 
tions this  probably  does  not  occur.  The  glycerin-phosphoric  acid 
is  subsequently  no  doubt  absorbed  together  with  some  of  the  fatty 
acids,  and  appears  in  the  urine  as  such.  The  cholin,  on  the  other 
hand,  is  further  decomposed,  with  the  formation  of  ammonia,  carbon 
dioxide,  and  methane. 

BACTERIAL    DECOMPOSITION    OF   THE    BILIARY   CON- 
STITUENTS. 

In  former  years  it  was  generally  supposed  that  the  biliary  acids 
after  their  elimination  into  the  intestinal  canal  were  there  absorbed 
to  a  large  extent  and  returned  to  the  liver,  while  a  smaller  portion 
was  decomposed  and  eliminated  in  the  feces.  Some  observers  even 
now  maintain  the  occurrence  of  such  a  circulation  of  the  bile-acids, 
but  there  is  a  strong  tendency  among  physiologists  at  present  to 
deny  its  existence.  As  a  matter  of  fact,  bile-acids  are  not  found  in 
the  blood  or  in  the  urine  under  normal  conditions. 

In  the  human  being,  moreover,  dyslysins  are  found  only  in  the 
feces,  while  the  amido-radicles  have  apparently  been  decomposed. 
In  other  animals  glycocholic  acid  has  been  found,  but  taurocholic 
acid  apparently  always  succumbs  to  the  action  of  the  bacteria. 
Of  the  fate  of  the  amido-radicles  we  know  little,  but  it  is  possible 
that  both  are  in  part  further  decomposed  and  in  part  absorbed. 
Taurin  may  then  appear  in  the  urine  either  as  such  or  as  tauro- 
carbaminic  acid;  but  it  may,  on  the  other  hand,  again  combine 
with  cholalic  acid  and  reappear  in  the  bile.  The  glycocoll  similarly 
may  in  part  be  transformed  into  urea ;  or  it  may  combine  with  the 


DECOMPOSITION  OF  THE  BILIARY  CONSTITUENTS.       203 

non-hydroxylated  aromatic  acids  which  are  also  formed  during  the 
process  of  intestinal  putrefaction,  and  appear  in  the  urine  as  hip- 
puric  acid  and  phenaceturic  acid;  or  it  may  find  its  way  to  the 
liver  and  be  re-eliminated  into  the  intestinal  tract  as  glycocholic 
acid. 

Bilirubin  is  reduced  to  hydrobilirubin  during  the  process  of 
intestinal  putrefaction  and  largely  eliminated  in  the  feces  as  such. 
This  reduction,  according  to  Nencki,  MacFadyen,  and  Sieber. 
occurs  in  man,  in  the  large  intestine.  A  portion,  however,  is  prob- 
ably absorbed  and  eliminated  in  the  urine  as  urobilin. 


CHAPTER    XI. 

THE  FECES. 

*  I  have  shown  in  the  preceding  chapters  that  the  greater  portion 
of  the  ingested  food  is  transformed  in  the  gastro-intestinal  canal 
into  material  which  can  be  utilized  by  the  body  for  purposes  of 
nutrition,  and  is  there  absorbed.  A  certain  proportion,  however, 
invariably  escapes  digestion,  and  is  partly  decomposed  by  the 
bacteria  of  the  intestinal  canal  into  the  various  substances  which 
have  been  considered  in  the  preceding  chapter.  These  substances 
in  turn  are  in  part  absorbed,  and  are  partly  eliminated  in  the  feces, 
together  with  particles  of  undigested  food  and  undigestible  material 
which  have  passed  through  the  digestive  tract  as  such.  In  addition 
we  find  here  the  various  native  and  decomposition -products  of  the 
bile,  the  pancreatic  juice,  the  enteric  juice,  in  so  far  as  they  have  not 
been  absorbed,  together  with  intestinal  mucus,  desquamated  epithe- 
lial cells,  and  bacteria. 

Consistence  and  Form. — The  consistence  and  form1  of  the  feces 
are  principally  dependent  upon  the  amount  of  water  that  is  present, 
and  vary  in  different  animals.  Generally  speaking,  they  are  softer 
in  the  herbivorous  animals  than  in  the  carnivora.  In  man  they 
usually  occur  in  the  characteristic  plastic,  cylindrical  form,  but  they 
may  at  times  be  mushy,  or  round  and  hard,  even  in  health. 

Amount. — The  amount  of  fecal  material  which  is  eliminated  in 
the  twenty-four  hours  depends  primarily  upon  the  amount  and  the 
character  of  the  food  that  has  been  ingested.  In  man  it  normally 
varies  between  100  and  200  grammes,  but  may  diminish  to  60 
grammes  or  rise  to  250  grammes,  even  in  health,  according  to  the 
preponderance  of  animal  food  or  of  vegetable  material,  which  has 
entered  into  the  composition  of  the  diet. 

Odor. — The  disagreeable  odor  of  the  feces  is  largely  due  to  indol 
and  skatol,  but  may  be  further  increased  by  the  presence  of  hydro- 
gen sulphide,  methane,  and  methyl-mercaptan. 

Color. — The  color  varies  with  the  character  of  the  food  ingested, 
and  is  usually  but  little  influenced  by  the  decomposition-products 
of  the  biliary  pigments.  In  carnivorous  animals  the  feces  are  almost 
black,  owing  to  the  presence  of  hsematin  and  sulphide  of  iron.  In 
adult  man  the  color  normally  varies  from  a  light  to  a  dark  brown. 
In  infants  in  which  the  bile-pigments  appear  as  such  the  feces  are 
of  a  bright-yellow  or  a  greenish-yellow  color. 

At  times  and  apparently  under  normal  conditions  stools  are  also 
passed  which  are  grayish  white  in  color  and  closely  resemble  the 
so-called  acholic  stools  which  are  observed  in  cases  of  biliary  ob- 

204 


GENERAL   CHEMICAL   COMPOSITION.  205 

struction.  Fats,  however,  are  not  necessarily  present  in  increased 
amounts,  and  there  is  no  reason  to  assume  that  the  biliary  passages 
are  not  patent  or  that  no  bile  is  being  secreted.  Possibly  the  lack 
of  color  in  such  stools  is  referable  to  the  formation  of  colorless 
decomposition-products  of  bilirubin,  such  as  the  leuko-urobilin  of 
Nencki,  and  in  the  last  instance  to  the  presence  in  the  intestinal 
canal  of  micro-organisms  which  are  usually  absent.  Nothing  defi- 
nite is,  however,  as  yet  known  of  the  conditions  which  favor  the  for- 
mation of  such  products. 

Macroscopic  Constituents. — On  macroscopic  examination  of  the 
feces  we  frequently  find  undigested  particles  of  food,  such  as  skins 
of  berries,  large  pieces  of  connective  tissue,  woody  vegetable  fibres, 
undigested  pieces  of  apples,  pears,  potatoes,  grains  of  corn,  flakes  of 
casein,  etc. 

Microscopic  Constituents. — On  microscopic  examination  we 
usually  find  undigested  bits  of  muscle-fibre,  connective-tissue  of  the 
white  fibrous  variety,  fragments  of  the  framework  of  vegetable 
matter,  often  still  enclosing  cells  with  starch-granules,  flakes  of 
casein,  globules  of  fat,  fatty  acid  needles,  crystals  of  calcium  oxalate, 
neutral  calcium  phosphate,  ammonio-magnesium  phosphate,  calcium 
lactate  (these  are  seen  especially  in  children  on  a  milk  diet),  and 
more  rarely  of  calcium  carbonate,  calcium  sulphate,  and  cholesterin. 
So-called  Charcot-Leyden  crystals,  which  consist  of  the  phosphate 
of  spermin,are  in  my  experience  only  found  under  pathological  con- 
ditions. We  further  meet  with  more  or  less  disintegrated  epithelial 
cells,  a  few  leucocvtes,  bits  of  mucus,  and,  above  all,  with  innumer- 
able micro-organisms.  Often,  indeed,  it  appears  as  though  the 
stools  consist  of  these  exclusively.  Their  number,  even  in  health, 
is  enormous.  Sucksdorff  thus  found  in  his  own  person  that  on  an 
average  53,124,000,000  were  eliminated  in    the  twenty-four  hours. 

Reaction. — In  adult  man  the  reaction  of  the  stools  is  usually 
alkaline,  sometimes  neutral,  and  but  rarely  acid.  Acid  stools,  on  the 
other  hand,  are  the  rule  in  infants. 

General  Chemical  Composition. — A  general  idea  of  the  average 
composition  of  the  human  feces  may  be  formed  from  the  following 
analyses,  which  are  taken  from  Gautier,  and  have  reference  to  1000 
parte  by  weight  of  the  fresh  material: 

Adult  man.  Snclding. 

Water      744.00  Ml.:; 

Solids      267.00  14S.7 

Total  organic  matter 208.75  137.1  ' 

Total  mineral  matter 10.95 2  13.6 

Alimentary  residue 84.00 

The  organic  material  yielded  ■ 

Aqueous  extract 53.40  53.5 

Alcoholic  extract      41.65  8.2 

Ethereal  extracl 30.70  17.0  » 

uding  -I  pari    "i  mucus,  epithelium,  and  calcareous  Milts. 
a  Not  ates. 

'  >!  this,     1  part 


206  THE  FECES. 

The  individual  constituents  of  the  feces  may  be  grouped  as 
follows  : 

1.  Food-material  which  has  escaped  the  process  of  digestion,  and 
of  bacterial  decomposition,  such  as  starches,  muscle-tissue,  connective- 
tissue,  fats,  etc. 

-.  Vndigestible  material,  which  lias  been  ingested  as  such,  or 
which  has  resulted  from  the  decomposition  of  complex  substances 
which  are  partly  digestible,  such  as  gums,  pectins,  resins,  ehitin, 
chlorophyl.  hamiatin.  and  insoluble  silicates,  sulphates,  phosphates, 
etc. 

o.  Derivatives  of  the  bile,  such  as  the  dyslvsins,  cholesterin,  and 
exceptionally  the  native  biliary  acids  as  such,  and  further  hydrobili- 
rubin,  stereobilin.  etc. 

4.  Intestinal  mucus. 

5,  Products  of  albuminous  digestion,  such  as  albumoses.  peptone- 
like bodies,  leuein,  ty  rosin,  asparaginic  acid,  and  glutaminie  acid. 

(i.  Products  of  bacterial  action.  These  comprise  the  entire  series 
of  tatty  acids  from  acetic  acid  to  palmitic  acid  ;  further,  lactic  acid, 
succinic  acid,  glutarie  acid,  leuein.  tyrosin,  hydroparaeumarie  acid, 
para-oxY-phenyl-aeetic  acid,  phenyl-propionic  acid,  phenyl-aeetie 
acid,  phenol,  paraeresol,  indol,  skatol,  skatol-earbonie  acid,  ammo- 
nium carbonate,  ammonium  sulphide,  etc. 

7.  Products  of  metabolism,  which  are  in  part  eliminated  through 
the  intestines,  such  as  uric  acid,  urea,  xanthin  bases,  etc. 

S.   Water. 

9.  Gases,  which  are  in  part  referable  to  the  various  fermentative 
and  putrefactive  processes  which  take  place  in  the  intestinal  canal, 
such  as  carbon  dioxide,  methane,  hydrogen,  hydrogen  sulphide, 
methvl-mereaptan,  and  phosphin.  The  nitrogen,  on  the  other  hand, 
which  is  also  constantly  met  with,  is  probably  derived  from  the 
blood,  and  has  in  part  been  swallowed. 

Many  of  these  substances  have  already  been  considered  in  detail, 
and  it  will  suffice  at  this  place  to  indicate  the  manner  in  which  the 
most  important  products  of  albuminous  putrefaction  can  be  isolated 
from  the  feces. 

ANALYSIS  OF  THE  PRODUCTS  OF  ALBUMINOUS 
PUTREFACTION. 

The  feces  are  diluted  with  water,  passed  through  a  muslin  filter 
to  remove  particles  of  food-material,  and  distilled  until  about  fonr- 
fifths  of  the  entire  volume  have  passed  over.  The  distillate  B  eon- 
tains  indol.  skatol,  phenol,  paraeresol,  and  the  volatile  acids  which 
are  present  in  the  free  state,  while  the  remaining  products  of  putre- 
faction are  found  in  the  residual  solution  A.  The  distillate  B  is 
neutralized  with  sodium  carbonate  and  redistilled.  This  second  dis- 
tillate. C,  contains  indol,  skatol,  phenol,  and  paraeresol,  while  the 
volatile    acids  remain  behind    as   sodium    salts,    and    can  be  sepa- 


PRODUCTS  OF  ALBUMINOUS  PUTREFACTION.  207 

rated  from  each  other  according  to  the  usual  analytical  methods 
(see  page  264).  Distillate  C  is  now  rendered  alkaline  with  sodium 
hydrate  and  extracted  with  ether  by  shaking.  This  takes  up  the 
indol  and  skatol,  which  may  be  obtained  in  crystalline  Conn  on 
evaporation  of  the  ether,  and  can  be  separated  from  each  other  by 
fractional  distillation  with  steam,  when  the  skatol  passes  into  the 
distillate  first.  The  residual  solution  of  C  contains  phenol  and 
paracresol  as  sodium  compounds.  This  is  now  acidified  with  sul- 
phuric acid  and  distilled,  when  the  phenols  pass  over,  and  may 
then   be  separated   from  each   other  as   described   elsewhere. 

The  residual  solution  A  is  now  concentrated  and  treated  with  a 
large  excess  of  alcohol,  in  order  to  precipitate  any  albumins  and 
mineral  salts  that  may  be  present.  The  alcoholic  filtrate  is  then 
transformed  into  an  aqueous  solution  and  aeiditied  with  sulphuric 
acid.  The  aromatic  acids  are  thus  set  free  and  are  extracted  with 
ether  by  shaking.  The  ethereal  solution  is  evaporated  to  dryness, 
the  residue  dissolved  in  a  small  amount  of  a  dilute  solution  of 
sodium  hydrate,  and  precipitated  with  barium  chloride.  The  fatty 
acids  are  thus  obtained  as  barium  soaps,  and  are  filtered  off".  They 
are  placed  in  water,  which  dissolves  the  salts  of  the  aromatic  acids. 
In  this  solution  the  acids  are  then  set  free  by  acidifying  with  sul- 
phuric acid,  and  are  extracted  with  ether.  The  ethereal  extract  is 
now  evaporated  to  dryness  and  the  free  acids  dissolved  in  water. 
On  distillation  in  a  current  of  steam,  phenyl-propionic  acid  and 
phenyl-acetic  acid  pass  over,  and  can  be  subsequently  separated 
from  each  other  by  fractional  distillation.  The  hydroxylated  oxy- 
acids  and  skatol-carbonic  acid  remain  behind.  The  latter  separates 
out,  on  further  concentration  of  the  solution  and  cooling,  in  the 
form  of  white  wart-like  granules,  while  the  oxy-acids  remain  behind, 
ami  can  be  separated  from  each  other  by  means  of  their  varying 
solubility  in  benzol.  They  can  be  recognized  by  applying  Millon's 
test.  Skatol-carbonic  acid,  on  the  other  hand,  reacts  in  very  much 
tin-  same  manner  with  yellow  nitric  acid  as  does  indol,  but  the  red 
color  is  in  this  ease  referable  to  a  different  pigment  (see  also  page 
204). 

Hydrobilirubin.  —  [t  has  been  stated  that  bilirubin  under  the 
influence  of  bacterial  action  supposedly  undergoes  a  process  of  reduc- 
tion in  the  intestinal  canal  and  is  transformed  into  hydrobilirubin. 
It  i-  assumed  that  as  such  it  is  then  in  part  absorbed  and  possibly 
appears  in  the  urine,  while  the  remaining  portion  is  eliminated  in 
the    feces.       A.CCOrding    to   some  observers,    it    is    identical    with    the 

etercobilin  of  Vanlair  and  Masius.  The  spectrum  of  the  two  bodies 
i-  very  similar,  but  while  solutions  of  hydrobilirubin  on  treatment 
with  chloride  of  zinc  and  ammonia  show  three  bands  of  absorption, 
stercobilin  i->  said  to  give  rise  to  four  bands.  Garrod  claims  that 
stercobilin  is  identical  with  the  urobilin  of  the  urine,  and  differs  from 
hydrobilirubin  in  containing  a  much  smaller  percentage  of  nitrogen, 
viz.,  1.1  I,  as  compared  with  \):l-l.     According  to  the  same  observer, 


208  THE  FECES. 

hydrobilirubin  is  a  laboratory  product,  and  is  met  with  neither  in 
the  feces  nor  the  urine. 

Excretin. — This  is  a  substance  which  was  first  isolated  by 
Marcet  from  the  feces  of  herbivorous  animals,  but  is  said  to  occur 
also  in  human  stools.  According  to  Hinterberger,  it  has  the  formula 
C20H36O,  and  is  thus  closely  related  to  cholesterin. 

Stercorin. — Stercorin,  or  serolin,  as  it  has  been  called,  is  a  sub- 
stance which  Flint  obtained  from  the  feces  of  man,  but  which  is 
probably  an  impure  form  of  cholesterin,  both  having  the  same 
general  reactions. 

MECONIUM. 

The  term  meconium  has  been  applied  to  the  material  which  accu- 
mulates in  the  intestinal  tract  during  foetal  life,  and  which  is  expelled 
soon  after  birth.  Food-products  are  here,  of  course,  wanting,  and 
as  the  intestinal  tract  of  the  foetus  is  free  from  bacteria  the  meconium 
consists  essentially  of  mucus,  desquamated  epithelial  cells,  and  the 
normal  biliary  constituents  which  are  present  in  the  intestinal  tract 
before  birth.  We  accordingly  find  bilirubin  and  biliverdin,  the 
former  often  in  crystalline  form,  the  native  biliary  acids,  a  small 
amount  of  fatty  acids,  cholesterin,  and  mineral  salts,  while  hydro- 
bilirubin, the  dyslysins,  leucin,  tyrosin,  indol,  skatol,  lactic  acid, 
albumoses,  etc.,  are  absent.  According  to  Zweifel,  it  contains  from 
79.8  to  80.5  per  cent,  of  water  and  from  19.5  to  20.2  per  cent,  of 
solids,  of  which  0.978  is  referable  to  mineral  ash,  0.797  to  choles- 
terin, and  0.772  to  fatty  acids. 

Its  color  is  a  dark  brownish-green,  and  the  reaction  usually  acid. 
In  general  appearance  it  resembles  pitch,  and  is  hence  also  spoken 
of  by  the  Germans  as  Kindspech  (infant  pitch). 


CHAPTER    XII. 

THE  URINE. 

The  urine  is  by  far  the  most  important  excretory  product  of  the 
animal  body,  and  the  medium  through  which  the  end-products  of 
nitrogenous  metabolism  and  soluble  mineral  salts  are  almost  exclu- 
sively eliminated  under  normal  conditions.  Abnormal  products  of 
metabolism  also,  and  many  substances  that  have  found  their  way 
into  the  circulation  from  without,  and  which  are  foreign  to  the  body, 
are  likewise  removed  in  this  manner,  either  as  such  or  in  a  more  or 
less  modified  form.  All  these  substances  are  found  in  the  urine  in 
aqueous  solution,  and  it  is  to  be  noted  that  of  the  total  amount  of 
water  which  is  daily  excreted  at  least  50  per  cent,  appears  in  this 
form. 

Formerly,  it  was  supposed  that  the  various  elements  which  are 
found  in  the  urine,  and  notably  the  mineral  salts  and  water,  were 
eliminated  by  a  simple  process  of  osmosis.  Later  it  was  shown, 
however,  that  in  the  elimination  of  the  organic  constituents  at  least 
the  renal  epithelium  of  the  uriniferous  tubules  plays  an  active  part, 
and  it  now  appears,  indeed,  that  all  the  substances  which  occur  in 
the  urine,  including  a  certain  amount  of  water  even,  are  removed 
from  the  blood,  viz.,  the  lymph,  through  the  intervention  of  the 
epithelial  cells.  It  is  supposed,  moreover,  that  these  structures 
possess  certain  selective  properties,  and  we  can  accordingly  under- 
stand why  the  composition  of  the  blood  always  remains  constant. 

The  kidneys  cannot  he  regarded  as  simple  excretory  organs,  how- 
ever, for  we  know  that  important  synthetic  processes  also  take  place 
in  them,  the  object  of  which  is  to  transform  certain  substances  which 
may  occur  in  the  circulating  blood  into  compounds  that  can  be  more 
readily  eliminated. 

The  most  important  synthesis  of  this  kind  is  that  of  glycocoll  and 
benzoic  acid,  which  results  in  the  formation  of  hippuric  acid  (see 
page  258). 

GENERAL  CHARACTERISTICS  OF  THE  URINE. 

The  general  appearance  of  the  urine  varies  in  different  animals. 
In  man  it  i-  perfectly  transparent  when  recently  passed,  but  soon 
becomes  turbid,  and  on  standing  deposits  a  light,  flocculent  sediment, 

which    consists   of  :i  mucinous    body   and    a   few    epithelial    cells   and 

leucocytes  thai  are  derived  from  the  urinary  passages. 

In  addition,  ;i  -mull  number  of  crystals  of  uric  acid  or  of  oxalate 
of  calcium  may  also  be  seen.     The  Bupernatanl   fluid   is  then  per- 

14  209 


210  THE   URINE. 

fectly  clear,  and  remains  so  if  care  is  taken  to  prevent  the  access  of 
micro-organisms.  If  left  exposed  to  the  air,  however,  bacterial 
decomposition  soon  takes  place.  Ammonia  appears  in  the  free  state, 
and  as  a  consequence  of  the  change  in  reaction  certain  constituents 
of  the  urine  are  precipitated  and  render  the  liquid  turbid.  Sooner 
or  later  they  settle  to  the  bottom,  but  owing  to  the  presence  of 
innumerable  micro-organisms  the  supernatant  fluid  remains  cloudy. 
Such  urine  is  said  to  have  undergone  ammoniaeal  decomposition. 

A  formation  of  sediments,  aside  from  the  light  cloud  which  de- 
velops in  every  urine  on  standing  for  a  short  while,  may,  however, 
also  occur  in  the  absence  of  micro-organisms.  In  the  winter-time  it 
is  a  common  experience  to  see  the  entire  volume  of  urine  become 
turbid  when  kept  in  a  cold  room.  This  is  owing  to  the  fact  that 
the  urates  of  the  urine  are  very  much  less  soluble  in  cold  than 
in  warm  water,  and  are  hence  thrown  down.  On  standing,  they 
soon  settle  to  the  bottom,  and  the  supernatant  liquid  remains  clear 
.so  long  as  bacterial  decomposition  does  not  occur.  A  similar  forma- 
tion of  sediments  is  observed  if  the  reaction  of  the  urine  is  alkaline, 
owing  to  the  presence  of  fixed  alkali  in  contradistinction  to  free 
ammonia.  This  may  at  times  be  observed  after  a  large  meal,  or 
after  the  administration  of  sufficiently  large  amounts  of  alkalies  as 
such,  or  of  substances  which  are  oxidized  to  alkaline  carbonates 
within  the  body.  In  such  an  event  the  urine  may  be  clear  when 
first  passed,  but  after  standing  a  short  time  it  becomes  turbid, 
and  deposits  a  sediment  of  phosphates  and  carbonates  of  the  alka- 
line earths.  The  change  is,  no  doubt,  due  to  an  escape  of  the 
carbon  dioxide  which  was  present  in  solution.  But  here  also  the 
supernatant  liquid  is  clear. 

In  herbivorous  animals,  by  which  an  alkaline  urine  is  passed 
habitually,  the  liquid  is  turbid  when  discharged.  In  man  the  pas- 
sage of  a  turbid  urine  is  always  abnormal,  excepting  during  the 
first  days  of  life,  when  cloudy  urine  is  the  rule.  This  is  largely 
referable  to  desquamated  epithelial  cells  and  relatively  large  amounts 
of  urates. 

While  the  urine  of  all  mammalian  animals  is  liquid,  the  lower 
animals  excrete  a  urine  that  is  more  or  less  solid.  In  birds 
and  reptiles,  for  example,  in  which  the  ureters  end  in  a  common 
cloaca  with  the  rectum,  the  excrements  appear  in  the  form  of  a  whit- 
ish  pasty  material.     A  gelatinous  urine  is  observed  in  turtles. 

The  color  of  the  urine  in  man  normally  varies  from  light  yellow 
to  dark  amber,  and  is  largely  influenced  by  the  concentration  of  the 
secretion  and  its  reaction.  Acid  urine  is  thus  always  darker  than  an 
alkaline  urine,  and  the  color  is  naturally  lighter  when  the  secretion 
is  abundant  than  when  scanty.  A  gradual  darkening  of  the  urine 
is  observed  when  the  material  is  kept  for  some  time  and  access  of 
micro-organisms  is  prevented. 

Deviation  from  the  normal  color  is  notably  observed  in  disease,  or 
following  the  administration  of  various  drugs,  but  may  also  occur  m 


GENERAL   CHARACTERISTICS  OF  THE   URIXE.  211 

apparently  healthy  individuals  in  consequence  of  certain  abnormali- 
ties of  metabolism  (see  page  261).  The  urine  may  then  be  of  a 
normal  color  when  recently  passed,  but  soon  darkens  on  standing 
and  finally  appears  almost  black.  In  diabetes  a  light  color  may  be 
associated  with  a  high  specific  gravity. 

The  odor  of  recently  passed  urine  is  peculiarly  aromatic,  and  is 
probably  referable  to  the  presence  of  several  volatile  acids.  Decom- 
posing urine  has  a  characteristic  odor,  which  is  in  part  due  to 
ammonia.  l 

Amount— The  amount  of  urine  eliminated  in  the  twenty-four 
hours  is  quite  variable  even  under  normal  conditions.  It  is  of 
course,  primarily  dependent  upon  the  amount  of  water  ingested,  but 
is  also  influenced  by  the  character  and  the  quantity  of  the  food  the 
process  of  digestion,  the  blood-pressure,  the  surrounding  tempera- 
ture the  emotions,  sleep,  exercise,  body-weight,  sex,  age,  etc!  It 
must  hence  differ  in  different  countries,  according  to  the  habits  of 
the  people,  the  climate,  etc.,  and  we  accordingly  find  that  different 
observers  give  different  figures.  In  Germany  and  Austria,  where 
much  beer  is  consumed,  from  1500  to  2000  c.c.  are  regarded  as 
average  amounts.  In  England  1000  to  1500  c.c.  are  regarded  as 
normal;  in  France,  1250  to  1300  c.c. 

In  this  country  I  have  found  that  the  average  daily  amount  is 

innnT  TonnVer'  and  am  T1^1  t0  reSard  an  elimination  of  from 
100  to  1200  c.c.^  as  normal  for  men,  while  in  women  a  somewhat 
smaller  quantity  is  normally  passed.     Children   pass   absolutely  less 

adults  m°re  UYme'  US  °0mparecl  with  their  body-weight,  than 

In  the  summer-time,  when  the  sweat-glands  are  especially  active 
and  when  larger  amounts  of  water  are  eliminated  through  the  lungs 

f'n  i  i  "^n  sccre;ion  of  urine  »  proportionately  less,  but  rarely 
tails  below  800  c.c.  unless  active  exercise  is  indulged  in  at  the  same 

During  repose,  moreover,  much  less  urine  is  voided  than  when 
exercise  is  taken  and  we  hence  find  a  smaller  secretion  of  urine 
during  the  night  than  during  the  day.  The  maximum  secretion  is 
usually  observed  a  few  hours  after  the  midday  meal 

ArtilH-.allv  .I,,,  secretion  can  be  increased  by  the  ingestion  of 
tnosearticles  of  food  which  tend  to  increase  the  blood-pressure  such 
as  coffee,  tea  and  alcohol.  Many  drugs  also  bring  about  the  'same 
effect,  though  the  modus  operandi  of  each  is  no,  known.  The  raosl 
important  medicinal  diuretics  are  digitalis,  squill,  broom,  juniper 
nitrous  ether,  urea,  etc.  Distilled  water  also  has  distinct  diuretic 
properties. 

In  disease,  and  notably  in  diabetes  mellitus,  diabetes  insipidus 
:i1"'  chronic  interstitial  nephritis  the  amount  of  urine  may  far  sur- 
1,;|"  ,,|r  nsnal  quantity,  and  may  indeed  exceed  10,000  c.c  in  the 
twenty-four  hours  (polyuria\  Abnormally  small  amounts,  on  the 
other  band  (oliguria),  are  observed  in  the  acute  febrile  diseases  in 


212  THE   URINE. 

various  diseases  of  the  circulatory  apparatus,  in  certain  diseases 
of  the  kidneys  and  liver,  etc.     Complete  anuria  may  indeed  occur. 

Specific  Gravity. — The  specific  gravity  of  the  total  amount 
passed  in  twenty-four  hours  normally  varies  between  1.015  and 
1.025.  Generally  speaking,  it  increases  with  the  solids,  the  amount 
of  water  remaining  the  same,  and  diminishes  as  the  amount  of  fluid 
increases  while  the  solids  remain  constant.  Under  pathological 
conditions,  however,  deviations  from  this  rule  are  not  uncommon. 
The  specific  gravity  may  then  fall  as  low  as  1.000  and  1.002,  or  may 
be  increased  to  1.050  and  even  higher. 

Reaction. — The  reaction  of  the  twenty-four  hours'  urine  is,  in 
man,  normally  acid,  sometimes  amphoteric,  and  more  rarely  alka- 
line. The  normal  acidity  is,  however,  not  due  to  the  presence  of  a 
free  acid,  but  to  acid  salts,  and  in  the  first  instance  to  acid  phos- 
phates. As  the  reaction  of  the  blood  is  alkaline,  the  question 
naturally  arises  :  How  is  it  that  an  acid  secretion  can  be  derived 
from  an  alkaline  fluid?  In  the  case  of  the  gastric  juice  we  have 
met  with  a  very  similar  phenomenon,  and  it  was  explained  that  the 
free  acid  in  that  case  most  likely  resulted  through  a  mass-action,  on 
the  part  of  carbonic  acid,  upon  sodium  chloride  within  the  oxyntic 
cells,  the  hydrochloric  acid  being  then  secreted  into  the  lumen  of 
the  glandular  ducts,  while  the  resulting  alkaline  carbonate  is 
returned  to  the  blood.  Similar  conditions  probably  exist  in  the 
kidneys,  where,  as  has  been  mentioned,  the  mineral  salts  are  also 
secreted  into  the  uriniferous  tubules  through  the  specific  activity  of 
the  renal  epithelial  cells.  AVe  may  imagine  that  here  also  a  mass- 
action  on  the  part  of  carbonic  acid  takes  place,  which  in  this  case, 
however,  is  directed  toward  the  alkaline  phosphates  of  the  blood,  as 
is  shown  in  the  equation  : 

Na2HP04  +  H2C03  =  NaH2P04  +  NaHC03. 

We  may  then  imagine  that  the  resulting  alkaline  carbonate  is 
returned  to  the  blood,  while  the  acid  phosphate  appears  in  the 
urine. 

The  acidity  of  the  urine,  however,  is  primarily  clue  to  the 
character  of  the  diet.  In  man  and  the  carnivorous  animals  this 
is  especially  rich  in  albumins,  and  contains  a  comparatively  small 
amount  of  alkaline  salts  or  of  organic  acids  which  could  be  trans- 
formed into  alkaline  carbonates  in  the  body.  During  the  process 
of  metabolism,  then,  the  ingested  albumins  are  broken  down,  and 
uric  acid,  hippuric  acid,  phenaceturic  acid,  oxalic  acid,  aromatic  oxy- 
acids,  and  notably  sulphuric  acid  and  phosphoric  acid  result.  These 
acids,  however,  are  immediately  transformed  into  neutral  salts  by 
combining  with  the  available  alkaline  carbonates  which  are  present 
in  the  lymph  and  the  blood.  As  a  consequence  the  alkalinity  of 
the  blood  must  of  necessity  diminish.  But  as  such  a  change  would 
give  rise  to  serious  disturbances,  and  as  there  is  a  strong  tendency 
on  the  part  of  the  body  to  maintain  the  composition  of  the  blood, 


GENERAL   CHARACTERISTICS  OF  THE   URINE.  213 

and  particularly  its  alkalinity,  constant,  a  loss  of  alkali  is  guarded 
against  by  subjecting  the  various  neutral  salts  to  the  specific  activity 
of  the  renal  epithelial  cells.  As  a  result  a  portion  of  the  alkali  is 
returned  to  the  blood,  and  acid  salts  hence  appear  in  the  urine. 

In  the  herbivorous  animals,  on  the  other  hand,  in  which  a  super- 
abundance of  alkaline  salts  is  either  directly  ingested  or  is  formed 
within  the  body  from  salts  of  organic  acids  which  have  been  taken 
with  the  food,  an  alkaline  urine  is  eliminated.  In  this  case  a 
formation  of  acid  salts  and  a  return  of  alkali  to  the  blood  are 
unnecessary.  Similar  conditions  at  times  occur  in  man,  and  the 
elimination  of  an  alkaline  urine,  the  alkalinity  being  due  to  a 
fixed  alkali,  cannot  hence  be  regarded  as  pathological.  During  the 
process  of  digestion,  indeed,  when  an  additional  amount  of  alkaline 
salts  finds  its  way  into  the  blood  in  consequence  of  the  formation 
of  hydrochloric  acid,  an  increased  alkalinity  would  result.  This, 
however,  is  prevented  by  the  excretion  of  a  urine  which,  if  not 
alkaline,  is  at  least  less  acid. 

It  has  been  stated  above  that  the  organic  acids  which  are  formed 
during  the  nitrogenous  metabolism  of  the  body  combine  with  the 
alkaline  carbonates  of  the  lymph  and  blood-plasma  to  form  neutral 
salts.  This  statement  requires  modification  in  so  far  as  it  conveys 
the  idea  that  the  acids  in  question  are  eliminated  in  the  urine  in 
combination  with  fixed  alkalies  only.  As  a  matter  of  fact,  this  is 
true  only  in  part,  and  a  certain  proportion  of  the  acid  is  eliminated 
in  combination   with  ammonia. 

Generally  speaking,  the  ammonium  salts  which  are  formed  within 
the  body  appear  in  the  urine  as  urea,  but  aside  from  their  impor- 
tance in  this  respect  they  represent  a  reserve  of  alkali  which  is  capa- 
ble of  preventing  an  undue  diminution  in  the  alkalinity  of  the  blood 
by  vicariously  taking  the  place  of  the  fixed  alkalies.  This  vicarious 
action  is  normally  also  at  work,  but  is  then  comparatively  insi<r- 
uificant  in  extent.  If,  however,  a  specially  large  demand  is  made 
upon  the  alkalies  of  the  body,  as  when  mineral  acids  are  ingested 
for  experimental  purposes,  the  vicarious  action  of  the  ammonium 
salts  :ii  one  enters  into  play.  Unless  carried  to  extremes,  the 
alkalinity  of  the  blood,  in  the  carnivorous  animals  at  least,  remains 
constant,  but  the  elimination  of  urea  i-  proportionately  less,  and  the 
deficif  of  nitrogen  in  this  form  appears  as  ammonia  in  combination 
with  acid-;. 

By  gradually  increasing  tlw  amount  of  acid  it  is  thus  possible  to 
bring  about    the  almosl    complete  disappearance  of  urea   from  the 

urine.     \    point,  however,  is  finally  reached  when  the  animal  suc- 
cumbs to  acid  intoxication,  and  then,  and  not  before,  may    free  acids 

appear  in  the   urine.     Death  in  such  cases  results  from  suffocation, 

a-  there  i-  not   Bufficienf  alkali  left   in  the  lymph  and  plasma  to  com- 
bine with  the  carbon  dioxide  in  the  tissues  (see  page  339). 

Conversely,  it  i-  possible  to  cause  the  ammonia  to  disappear  from 
the  urine  by  the  aaminisf  rat  ion  of  a  sufficiently  large  quantity  of 


214  THE   URINE. 

alkali,  and  as  a  consequence  an  increase  in  the  amount  of  urea 
occurs  directly  proportionate  to  the  amount  of  ammonia  formerly 
present.  In  herbivorous  animals,  in  which  such  a  vicarious  action 
is  never  necessary  under  normal  conditions,  it  is  accordingly  but 
little  developed,  and  they  hence  soon  die  even  after  the  administra- 
tion of  comparatively  small  amounts  of  mineral  acids. 

It  has  been  stated  that  the  acid  reaction  of  the  urine  is  essentially 
due  to  the  presence  of  acid  phosphates.  Besides  the  acid  phosphates 
normal  urine,  however,  contains  a  certain  amount  of  neutral  phos- 
phates, and  it  may  happen  that  both  are  present  in  equal  proportion. 
But  as  the  neutral  phosphates  show  an  alkaline  reaction,  a  neutral 
point  cannot  be  reached,  and  such  urines  hence  color  red  litmus- 
paper  blue,  and  the  blue  paper  red ;  in  other  words,  they  are  ampho- 
teric. Such  a  reaction  is  not  infrequently  observed,  but  is,  of  course, 
an  accidental  occurrence. 

When  allowed  to  stand  exposed  to  the  air,  every  urine  undergoes 
ammoniacal  decomposition.  This  is  owing  to  the  action  of  certain 
micro-organisms  upon  urea,  which  is  decomposed,  with  the  forma- 
tion of  ammonia,  water,  and  carbon  dioxide,  as  shown  in  the 
equations : 

(1)  CO(NH2)2  +  2H20  =  (NH4)2C03. 

(2)  (NH4)2C03  =  2NH3  +  H20  +  C02. 

The  action  is  thus  a  hydrolytic  decomposition,  and  is  referable  to 
the  activity  of  a  special  ferment,  which  is  found  in  the  micro-organ- 
isms in  question,  notably  the  Micrococcus  urea?  and  the  Bacterium 
urea?. 

As  a  result  of  the  presence  of  free  ammonia,  the  soluble  phosphates 
of  the  alkaline  earths  are  then  precipitated  as  tricalcium  phosphate 
and  as  ammonio-magnesium  phosphate,  and  the  soluble  urates  are  at 
the  same  time  transformed  into  the  insoluble  ammonium  salt. 

At  times  an  increase  in  the  acidity  of  the  urine  is  observed  on 
standing,  and  is  generally  ascribed  to  a  peculiar  acid  fermentation 
of  contained  alcohol,  traces  of  carbohydrates,  and  the  like.  More 
often,  however,  a  decrease  in  the  acidity  occurs,  even  though  micro- 
organisms are  absent.  This  is  owing  to  a  decomposition  of  neutral 
urates  by  the  acid  phosphate  of  sodium.  Acid  urates  thus  result, 
and  may  be  further  decomposed,  with  the  liberation  of  uric  acid. 
Both  urates  and  uric  acid  are  then  thrown  down  in  consequence  of 
the  diminished  acidity  of  the  fluid,  and  they  are  hence  no  longer 
capable  of  influencing  the  reaction.  The  changes  which  here  take 
place  may  be  represented  by  the  equations  : 

(1)  NaH2P04        +  C5H2Na2N403  =  Na2HP04  +  C5H3NaN403. 

(2)  C5H3NaN403  +  NaH2P04        =  Na2HP04  +  C5H4N403. 

As  the  reaction  of  the  urine  is  dependent  in  the  first  instance  upon 
the  character  and  the  quantity  of  the  food  ingested,  viz.,  the  amount 


GENERAL   CHARACTERISTICS  OF  THE   URINE.  215 

of  albumins  and  alkaline  salts  present,  or  of  salts  which  can  be  trans- 
formed within  the  body  into  alkaline  carbonates,  it  follows  that  a 
highly  acid  urine  must  also  result  when  an  increased  destruction  of 
tissue-albumins  is  taking  place  from  whatever  cause.  "We  accord- 
ingly find  a  very  acid  urine  in  various  pathological  conditions,  nota- 
bly in  fevers. 

An  alkaline  urine  will  similarly  result  when,  as  in  pneumonia  and 
in  diseases  in  which  large  accumulations  of  fluid  occur  in  the 
serous  cavities  of  the  body,  and  where  a  certain  amount  of  alka- 
line salts  has  thus  been  withdrawn  from  the  circulation,  absorption 
subsequently  occurs.  Alkaline  salts  are,  however,  retained  from 
the  ingested  food,  and  an  increased  elimination  occurs  when  the  addi- 
tional supply  finds  its  way  into  the  plasma  from  these  various  sources. 
A  notable  change  in  the  normal  alkalinity  of  the  blood  can  hence 
scarcely  occur  so  long  as  a  sufficient  amount  of  alkali  is  furnished  in 
the  food. 

In  order  to  decide  whether  the  alkaline  reaction  of  a  specimen  of 
urine  is  due  to  the  presence  of  fixed  or  volatile  alkali,  a  strip  of  red 
litmus-paper  is  clamped  in  the  cork  of  the  bottle,  and  so  arranged  as 
not  to  touch  the  liquid.  If  free  ammonia  is  present,  the  red  color 
changes  to  blue,  while  fixed  alkali  is  indicated  only  when  the  paper 
comes  into  contact  with  the  urine. 

Determination  of  the  Acidity  of  the  Urine. — As  the  acidity  of  the 
urine  is  almost  exclusively  due  to  the  presence  of  acid  phosphates,  its 
determination  resolves  itself  into  the  estimation  of  these  salts.  The 
resulting  values  are  expressed  in  terms  of  hydrochloric  acid,  of  which 
102.-S  mgrms.  correspond  to  100  mgrms.  of  the  diacid  sodium  salt. 
Negative  values  are  similarly  expressed  in  terms  of  sodium 
hydrate. 

Freuxd's  Method. — The  total  amount  of  phosphoric  acid  is  first 
determined  as  described  on  page  220).  In  a  second  portion  the 
monacid  phosphates  are  then  estimated  as  follows:  50  c.c.  of 
urine  are  precipitated  with  a  normal  solution  of  barium  chloride,  10 
c.c.  being  added  for  every  100  mgrms.  of  the  total  amount  of  phos- 
phoric acid  that  has  been  found.  The  mixture  is  diluted  with  water 
to  100  c.c,  filtered,  and  the  phosphoric  acid  determined  in  50  c.c.  of 
the  filtrate,  lint  as  barium  chloride  precipitates  not  only  the  mon- 
acid phosphate,  but  also  a  .-mall  amounl  of  the  normal  phosphates, 
with  the  simultaneous  formation  of  a  small  amount  of  diacid  phos- 
phate-, which  latter  pass  into  solution,  an  error  i<  thus  incurred. 
This,  however,  remain-  constant,  and  amounts  to  :;  per  cent. in  favor 
of  the  diacid  phosphates.  It  is  deducted  from  the  latter,  and  the 
total  amounl  of  acia  -alt-  is  then  determined  by  calculation.  The 
result   i-  expressed  in  terms  of  hydrochloric  acid. 

If  relative  value-,  on    the   other  hand,  are  desired,  the  percentage 

of  the  diacid  -alt-  i-  ascertained  and  compared  with  the  total  amount 
of  phosphoric  acid,  a-  shown  in  the  following  example : 

The    total    amount  of  urine    i-    2000  C.C.,  and    1  he  total  amount  of 


216  THE   URINE. 

phosphoric  acid  in  terms  of  P2Os  is  7.72  grammes,  while  the  corre- 
sponding amount  of  acid  phosphates  is  6.736  grammes  after  correct- 
ing as  above  indicated.  The  percentage  of  the  acid  phosphates,  as 
compared  with  the  total  P205,  would  then  be  87.2,  as  is  seen  from 
the  calculation  : 

7.72  :  100  :  :  6.736  :  x. 

Chemical  Composition  of  the  Urine. — A  general  idea  of  the 
chemical  composition  of  the  urine  and  the  quantitative  variations  of 
the  individual  components  may  be  formed  from  the  accompanying 
table,  which  I  have  constructed  from  numerous  analyses  made  in 
my  laboratory.  The  individuals  from  which  the  urine  was  obtained 
were  all  adults,  and  in  their  general  mode  of  life,  as  regards  diet, 
exercise,  etc.,  followed  the  common  habits  of  the  average  American 
city-dweller. 

Analysis  of  Urine. 

Water 1200-1700  grammes. 

Solids 60.0 

Inorganic  solids 25.0  -26.0         " 

Sulphuric  acid  (H2S04) 2.0-2.5 

Phosphoric  acid  (P205) 2.5-3.5         " 

Chlorine  (NaCl) .    .   '.  10.0  -15.0         " 

Potassium  (K2Oj 3.3 

Calcium  (CaO) 0.2-0.4 

Magnesium  (MgO) 0.5 

Ammonia  (NH3) 0.7 

Fluorides,  nitrates,  etc 0.2 

Organic  solids 20.0  -35.0 

Urea 20.0  -30.0         " 

Uric  acid 0.2-1.0         " 

Xanthin  bases 1.0 

Kreatinin 0.05-  0.08        « 

Oxalic  acid 0.05 

Conjugate  sulphates 0.12-  0.25       " 

Hippuric  acid 0.65-  0.7 

Volatile  fatty  acid 0.05 

Other  organic  solids 2.5 

THE  INORGANIC  CONSTITUENTS  OF  THE   URINE. 

The  inorganic  constituents  of  the  urine  represent  the  excess  of 
mineral  salts  which  find  their  way  into  the  blood  from  the  digestive 
tract,  or  which  develop  within  the  body  during  the  decomposition 
of  the  albumins.  As  has  been  indicated,  they  are  eliminated 
through  the  specific  activity  of  the  renal  epithelial  cells,  so  that 
the  composition  of  the  blood  always  remains  constant.  We  accord- 
ingly find  that  the  ingestion  of  iarge  amounts  of  food  invariably 
leads  to  an  increased  elimination  of  salts,  and  that  conversely 
smaller  amounts  are  excreted  when  smaller  amounts  are  ingested. 
This  is  true  more  especially  of  the  chlorides  and  the  phosphates, 
while  the  sulphates  are  largely  referable  to  albuminous  destruction, 
and  are  only  ingested  as  such  in  minimal  quantities.  As  the  chlo- 
rides, moreover,  are  far  more  abundant  in  food-stuifs  than  the  phos- 


THE  INORGANIC  CONSTITUENTS  OF  THE   URINE.         217 

phates,  any  variations  from  the  normal  will  affect  these  particularly. 
Under  various  pathological  conditions,  where  a  deficient  amount  of 
food  and  of  inorganic  salts  is  ingested,  or  where  a  considerable 
amount  of  the  salts  is  removed  from  the  circulation,  as  in  conse- 
quence of  hemorrhages,  the  formation  of  exudates  and  transudates, 
etc.,  smaller  amounts  are  accordingly  eliminated,  and  it  may  happen, 
indeed,  that  the  chlorides  disappear  from  the  urine  altogether.  It 
is  noteworthy,  moreover,  that  an  arrest  in  the  elimination  of  the 
chlorides  may  then  also  occur  even  though  a  fair  amount  of  salt  is 
introduced  with  the  food.  In  such  an  event  we  must  assume  that  a 
retention  is  taking  place  in  the  body,  in  consequence  of  the  fact  that 
the  lost  fluid,  with  its  various  inorganic  constituents,  is  gradually 
being  replaced.  Subsequently,  when  absorption  of  an  exudate  or  a 
transudate  takes  place,  the  inorganic  solids  find  their  way  into  the  gene- 
ral circulation,  but  are  at  once  eliminated,  as  they  are  present  in  excess. 

The  phosphates  and  sulphates  are  likewise  diminished  under  the 
conditions  just  mentioned,  but  do  not  disappear  entirely,  as  they  are 
in  part  derived  from  the  albumins,  which  are  constantly  undergoing 
destruction  during  the  nitrogenous  metabolism  of  the  body.  The 
diminution  in  the  amount  of  the  phosphates,  however,  exceeds  that 
of  the  sulphates,  as  a  small  fraction  only  of  the  former  is  due  to 
this  source,  while  the  latter  are  largely  derived  from  the  disinte- 
grated albumins. 

The  tenacity  with  which  the  body  maintains  the  normal  composi- 
tion of  the  blood  is  also  well  shown  if  the  chlorides  are  gradually 
diminished  in  the  food,  and  if  their  elimination  from  the  blood  is 
stimulated  by  the  copious  ingestion  of  diuretics.  A  point  is  soon 
reached  when  the  salt  in  question  no  longer  appears  in  the  urine, 
beyond  traces.  If  at  this  stage  the  blood  is  examined,  it  will 
be  found  that  the  amount  of  chlorides  is  practically  the  same  as 
under  normal  conditions.  There  is  a  limit  to  this  power  of  re- 
taining the  mineral  salts,  however,  and  if  the  chlorides  are  withheld 
for  a  length  of  time  and  diuresis  remains  active,  a  gradual  loss 
occurs  nevertheless,  and  in  time  results  in  the  death  of  the  animal. 
It  appears,  however,  that  it  is  not  the  loss  of  chlorine  which  the 
body  tends  to  prevent,  but  that  the  sodium  is  the  component  which 
i-  of  prime  importance.  This  becomes  apparent  when  the  potassium 
salt  i-  substituted  for  the  sodium  compound,  when  the  same  reten- 
tion of  sodium  chloride  occurs,  while  the  potassium  salt  is  eliminated 
in  the  urine  In  this  case,  also,  death  ultimately  results  from  what 
is  very  improperly  termed  "  chlorine-hunger." 

If. -it  the  atage  when  the  chlorides  have  practically  disappeared 
from  the  urine  sail  i-  added  to  the  diet,  a  partial  retention  of  this 
occur-  until  the  original  equilibrium  has  been  restored.  After  that 
a  normal  elimination  is  again  observed,  and  the  amount  then  cx- 
creted   practically  corresponds  to  the  quantity  ingested. 

These  remark-  also  hold  good  for  the  phosphates  and  sulphates  of 
the  body,  though  with   certain  restrictions. 


218  THE   URINE. 

The  bases  which  are  found  in  the  urine  in  combination  with  hydro- 
chloric acid,  sulphuric  acid,  and  phosphoric  acid,  are  sodium,  potas- 
sium, calcium,  magnesium,  and  ammonium.  The  latter,  however, 
occurs  only  in  the  urine  of  man  and  carnivorous  animals.  Calcium 
and  magnesium  occur  almost  exclusively  as  phosphates,  both  of  the 
monacid  and  the  diacid  type.  Traces,  however,  no  doubt  exist  in 
combination  with  hydrochloric  acid  and  sulphuric  acid  as  well,  but 
the  greater  portion  of  these  two  acids,  as  also  of  the  phosphoric  acid, 
is  found  in  the  form  of  sodium  and  potassium  salts.  The  ratio 
between  the  two  latter  is  usually  placed  at  3  : 5,  in  favor  of  sodium. 
It  is  believed  that  the  monacid  phosphates  of  the  alkaline  earths, 
and  notably  the  calcium  salts,  are  held  in  solution  owing  to  the 
presence  of  sodium  chloride  and  the  diacid  phosphates  of  the  alkalies,, 
to  which  the  acidity  of  the  urine  is  due. 

The  alkaline  phosphates  normally  exceed  the  earthy  phosphates 
by  one-third,  and  it  is  to  be  noted  that  the  latter  are,  in  part  at 
least,  also  eliminated  by  the  mucous  membrane  of  the  large  intes- 
tine. It  consequently  follows  that  estimation  of  the  ratio  between 
the  two  forms  of  phosphates  can  only  be  of  value  when  the  amount 
which  is  thus  excreted  is  known.  Practically,  such  a  determination 
is,  however,  impossible,  as  a  variable  amount  of  earthy  phosphates 
— in  fact,  the  greater  portion  of  that  wrhich  has  ~ been  ingested — is 
directly  eliminated  through  this  channel. 

While  the  greater  portion  of  the  sulphuric  acid  which  results  from 
the  destruction  of  albumins  within  the  tissues  of  the  body  is  found 
in  the  urine  in  combination  with  inorganic  bases  only,  a  variable 
fraction  also  occurs  united  with  certain  aromatic  substances  which 
are  formed  during  intestinal  putrefaction.  The  resulting  bodies  are 
spoken  of  as  conjugate  or  ethereal  sulphates,  and  normally  repre- 
sent about  one-tenth  of  the  total  amount  of  sulphuric  acid  that 
appears  in  the  urine.  They  comprise  the  alkaline  salts  of  phenol, 
indoxyl,  and  skatoxyl,  and  will  be  considered  later. 

The  mineral  and  conjugate  sulphates  together  are  spoken  of  as" 
the  "  acid  "  sulphur  of  the  urine,  in  contradistinction  to  the  so-called 
neutral  sulphur,  which  represents  a  variable  fraction  that  escapes 
oxidation  in  the  body  and  finds  its  way  into  the  urine  as  such.  This 
comprises  such  substances  as  thiosulphuric  acid,  tauro-carbaminic 
acid,  sulphocyanic  acid,  cystin,  cystein,  ethyl  sulphide,  etc.  They 
are  described  in  detail  at  another  place. 

In  addition  to  the  salts  mentioned,  a  variable  amount  of  carbo- 
nates is  found  in  every  urine.  In  man  and  the  carnivorous  ani- 
mals this  is  usually  small ;  but  in  the  herbivorous  animals  large 
quantities  are  normally  found,  and  the  alkaline  reaction  of  such 
urine  is  indeed  largely  referable  to  this  source.  The  acid  occurs  in 
combination  with  the  alkalies  and  the  alkaline  earths,  and  owing  to 
the  presence  of  the  latter  especially  the  urine  of  such  animals  is 
normally  turbid. 


THE  INORGANIC  CONSTITUENTS  OF  THE   URINE.         219 

Of  other  inorganic  constituents,  every  urine  also  contains  iron 
(partly  in  organic  combination),  silicates,  fluorides,  hydrogen  per- 
oxide, and  nitrates,  all  of  which,  however,  are  present  only  in 
traces.  The  nitrates  are  probably  introduced  with  vegetable  food, 
and  disappear  from  the  urine  during  starvation.  During  amino- 
niacal  fermentation  they  arc  reduced  to  nitrites,  and  later  disappear. 

The  quantitative  variations  of  the  inorganic  constituents  of  human 
urine  are  shown  in  the  following  table  : 

Chlorides  (calculated  as  IIC1) 6.2-9.4  grammes. 

Phosphates  (calculated  as   P.,05) 2.5-3.0         " 

Sulphates  (calculated  as  H2S04) 2.0-2.5         " 

Sodium    (calculated    as   iSa./J) 4.0-6.0         " 

Potassium  (calculated  as  K20) 2.0-3.0         " 

Ammonium  (calculated  as  NH3) 0.7     gramme. 

Magnesium  (calculated  as  MgO) 0.5-0.0         " 

Calcium  (calculated  as  CaO)      0.2-0.4 

Quantitative  Estimation  of  the  Mineral  Ash. 

Ten  c.c.  of  urine  are  placed  in  a  weighed  crucible  and  evaporated 
at  a  temperature  of  about  100°  C.  The  crucible  is  then  covered 
with  its  lid  and  carefully  heated  over  a  small  flame  until  the  organic 
matter  has  been  carbonized  and  fumes  are  no  longer  evolved.  On 
cooling,  the  residue  is  extracted  with  boiling  water.  The  washings 
are  passed  through  a  small  filter,  the  weight  of  the  ash  of  which  is 
known,  and  the  filter,  together  with  the  carbonaceous  residue,  is 
incinerated  until  a  white  ash  is  obtained.  This  process  may  be 
aided,  if  necessary,  by  moistening  the  material  with  a  little  alcohol 
or  water.  The  washings  are  then  placed  in  the  crucible  and  evapo- 
rated at  100°  C.  The  residue  is  finally  dried  in  a  hot-air  bath, 
heated  until  the  bottom  of  the  crucible  just  turns  red,  and  is  then 
allowed  to  cool  over  sulphuric  acid  and  weighed.  The  weight  of  the 
mineral  ash  of  the  10  c.c.  of  urine  is  then  ascertained  by  deducting 
that  of  the  crucible  and  the  ash  of  the  filter. 

Quantitative  Estimation  of  the  Chlorides. 

The  chlorides  of  the  urine  are  most  conveniently  estimated  accord- 
ing  to  the  method  of  Salkow.-ki- Yolhard.  To  this  end,  10  c.c.  of 
urine  an:  diluted  with  50  C.C.  of  distilled  water,  and  treated  with  4 
c.c.  of  concentrated  nitric  acid  and  15  c.c.  of  a  standard  solution  of 
silver  nitrate.  The  mixture  is  further  diluted  to  1 00  c.c,  thor- 
oughly agitated,  and  passed  through  a  dry  filter.  In  a  carefully 
measured  portion  of  the  filtrate  die  excess  of  silver  is  then  titrated 
with  a  solution  of  potassium  sulphocyanide  of  such  strength  that  25 
c.c.  correspond  to  lo  c.c.  of  the  silver  solution.  A  few  drop- of  a 
saturated  solution  of  amraonio-ferric  alum  serve  as  indicator.  The 
amount  of  silver  solution  used  in  the  precipitation  of  the  chlorides 
in  the  10  c.c.  of  urine  is  then  calculated.  The  number  of  cubic 
centimeter*  which   wa-   necessary  for  this   purpose,  multiplied  by 


220  THE   URINE. 

0.01,  indicates  the  amount  of  chlorides  present  in  the   10  c.c.  of 
urine,  calculated  as  sodium  salt. 

The  presence  of  albumins  and  sugar  does  not  interfere  with  the 
method. 

Quantitative  Estimation  of  the  Phosphates. 

To  determine  the  amount  of  the  alkaline  and  earthy  phosphates 
together,  50  c.c.  of  urine  are  treated  with  5  c.c.  of  a  solution  con- 
taining about  100  grammes  of  sodium  acetate  and  100  c.c.  of  a  30 
per  cent,  solution  of  acetic  acid  to  the  liter.  In  this  manner  any 
monacid  phosphates  that  may  be  present  are  transformed  into  diacid 
^phosphates.  A  few  drops  of  tincture  of  cochineal  are  then  added, 
and  the  mixture  heated  to  the  boiling-point  and  titrated  with  a 
standard  solution  of  uranyl  acetate  or  nitrate  until  a  greenish  color 
is  noticed  in  the  resulting  precipitate  of  uranyl  phosphate  which 
does  not  disappear  on  stirring.  From  the  number  of  cubic  centim- 
eters employed  the  corresponding  amount  of  phosphates  is  then 
determined  in  terms  of  P2Os.  The  uranium  solution  is  of  such 
strength  that  20  c.c.  represent  0.1  gramme  of  P205. 

The  presence  of  sugar  and  albumins  does  not  interfere  with  the 
method. 

Separate  Estimation  of  the  Earthy  and  Alkaline  Phosphates. 

Two  hundred  c.c.  of  urine  are  rendered  strongly  alkaline  with 
ammonia  and  set  aside  for  several  hours.  The  earthy  phosphates 
are  thus  precipitated,  and  are  collected  on  a  small  filter,  washed  with 
dilute  ammonia  (1  :  3),  transferred  to  a  beaker,  and  dissolved  with 
as  little  acetic  acid  as  possible.  Distilled  water  is  added  so  as  to 
make  the  volume  of  the  liquid  about  50  c.c,  when  the  solution  is 
boiled  and  titrated  as  above.  In  a  second  portion  of  the  urine 
the  total  amount  of  phosphates  is  then  determined.  The  difference 
between  the  two  results  indicates  the  amount  of  phosjjhates  which 
is  present  in  combination  with  alkalies. 

If  it  is  desired  to  remove  the  total  phosphates  from  a  specimen 
of  urine  preliminary  to  some  further  step  in  analysis,  the  fluid  is 
rendered  alkaline  with  the  hydrate  of  an  alkaline  earth  and  pre- 
cipitated with  a  soluble  calcium  or  barium  salt.  Or  we  may  pre- 
cipitate directly  with  neutral  or  basic  acetate  of  lead.  In  the  first 
instance,  the  excess  of  calcium  or  barium,  and  in  the  second,  that 
of  lead,  must  then  be  removed. 

Quantitative  Estimation  of  the  Sulphates. 

To  determine  the  amount  of  both  mineral  and  conjugate  sulphates, 
100  c.c.  of  urine  are  treated  with  8  c.c.  of  strong  hydrochloric  acid 
and  heated  to  the  boiling-point.     In  this  manner  the  conjugate  sul- 


THE   ORGANIC  CONSTITUENTS  OF  THE   URINE.  221 

pbates  are  decomposed,  with  the  liberation  of  the  sulphuric  acid, 
which  is  then  precipitated,  together  with  the  mineral  sulphates,  as 
barium  sulphate.  To  this  end,  20  c.c.  of  a  hot  saturated  solution  of 
barium  chloride  are  added  to  the  hot  liquid.  The  mixture  is  kept 
on  a  boiling  water- bath  until  the  precipitate  has  settled,  when  it  is 
collected  on  a  small  filter,  washed  with  boiling  water,  then  with  hot 
alcohol,  and  finally  with  ether.  After  incineration  the  filter  ash  is 
deducted  from  the  total  weight.  The  result  may  be  expressed  in 
terms  of  H.,S04,  of  SOa  or  S,  by  multiplying  the  weight  of  the  barium 
sulphate  by  0.42015,  6.34301,  or  0.137*44,  respectively. 

To  determine  the  amount  of  mineral  sulphates  and  of  conjugate 
sulphates  separately,  100  c.c.  of  urine  are  treated  with  an  equal 
volume  of  an  alkaline  solution  of  barium  chloride,  which  consists  of 
two  volumes  of  a  solution  of  barium  hydrate  and  one  volume  of  the 
chloride,  both  saturated  at  ordinary  temperatures.  The  mineral  sul- 
phates are  thus  precipitated  together  with  the  phosphates  and  are 
filtered  off.  One  hundred  c.c.  of  the  filtrate,  corresponding  to  50 
c.c.  of  the  urine,  are  now  strongly  acidified  with  hydrochloric  acid 
and  boiled.  The  conjugate  sulphates  are  decomposed,  and  the  lib- 
erated sulphuric  acid  is  thrown  down,  as  above.  The  process  is 
then  continued  as  described.  The  resulting  value  represents  the 
amount  of  sulphuric  acid  which  was  present  in  combination  with 
phenol,  indoxyl,  and  skatoxyl.  By  deducting  this  value  from  the 
total  amount  of  sulphuric  acid  the  mineral  portion  is  ascertained. 

Test  for  Nitrates. — To  demonstrate  the  presence  of  nitrates, 
200  c.c.  of  urine  are  treated  with  30  to  40  c.c.  of  chemically  pure, 
concentrated  sulphuric  acid  or  hydrochloric  acid,  and  distilled  upon 
a  sand-bath.  The  distillate  is  received  into  a  dilute  solution  of 
caustic  alkali.  Owing  to  the  presence  of  reducing  substances  in  the 
urine,  the  nitric  acid  is  thus  transformed  into  nitrous  acid,  and 
passes  over  as  such.  The  presence  of  nitrites  may  then  be  demon- 
strated as  usual. 

THE  ORGANIC  CONSTITUENTS  OF  THE  URINE. 

The  organic  constituents  of  the  urine  comprise  the  normal  end- 
products  of  the  nitrogenous  metabolism  of  the  body,  various  products 
of  albuminous  putrefaction  which  have  found  their  way  into  the 
general  circulation  from  the  intestinal  canal,  and  certain  pigments 
which  are  more  or  les>  intimately  related  to  the  normal  blood-pig- 
ment. In  addition,  traces  of  various  other  substances  may  be 
encountered,  the  origin  of  which  is  obscure.     Under  pathological 

Conditions  we  meet  with  certain  normal  constituents  of  the  blood 
which    generally  do    not    appear    in    the    urine   as   such,  or   occur    in 

infinitesimally  small  amount-,  and  also  with  various  abnormal 
products  of  metabolism,  all  of  which  will  be  considered  in  detail. 


222  THE    URINE. 

The  Nitrogenous  Constituents  of  the  Urine. 

Urea. 

While  in  birds  and  reptiles  the  greater  portion  of  the  urinary 
nitrogen  is  excreted  in  the  form  of  uric  acid,  urea  constitutes  the 
most  important  end-product  of  the  nitrogenous  metabolism  of  the 
remaining  groups  of  vertebrate  animals.  In  man,  86  per  cent,  of  the 
total  nitrogen  eliminated  in  the  urine  appears  in  this  form. 

Origin. — Formerly  it  was  supposed  that  urea  resulted  from  uric 
acid  through  a  process  of  oxidation,  and  that  this  was  its  only 
source.  We  have  seen  that  the  formation  of  urea  from  uric  acid  is 
possible,  and  we  cannot  deny  that  a  certain  proportion  of  the  sub- 
stance may  be  derived  in  this  manner.  Modern  researches,  how- 
ever, have  shown  that  in  man  and  the  mammalian  animals  uric  acid 
is  largely  derived  from  a  destruction  of  the  nucleins  within  the 
body,  and  results  from  oxidation  of  the  xanthin  bases,  which  are 
thus  set  free.  In  birds  and  reptiles,  on  the  other  hand,  the  greater 
portion  of  the  uric  acid  is  formed  synthetically  from  simpler  sub- 
stances, and  is  hence  not  directly  comparable  to  the  form  which 
is  found  in  the  higher  animals.  In  these  a  synthetic  formation  is 
also  possible,  but  probably  does  not  occur  under  normal  conditions. 
As  we  can  therefore  recognize  one  origin  of  uric  acid  only  in  the 
mammal,  and  as  this  source  of  the  nitrogen  is  insignificant  when 
compared  with  the  large  amount  of  urea  actually  found,  we  are 
forced  to  the  conclusion  that  the  greater  portion  of  the  urea  must 
originate  in  a  different  Avay. 

It  has  been  repeatedly  shown  that  during  the  decomposition  of 
the  albumins  by  means  of  acids  and  alkalies,  as  also  during  the 
process  of  tryptic  digestion  and  albuminous  putrefaction,  a  large 
amount  of  mono-amido-acids  results.  It  has  hence  been  supposed 
that  these  bodies  probably  represent  intermediary  products  in  the 
transformation  of  the  albuminous  nitrogen  into  urea,  and  it  has 
actually  been  demonstrated  that  in  mammals — and  to  these  I  shall 
confine  my  remarks  for  the  present — the  administration  of  such 
acids  in  the  food  is  followed  by  a  corresponding  increase  in  the 
amount  of  urea.  Under  certain  pathological  conditions,  moreover, 
these  acids  appear  in  the  urine  as  such,  and  it  is  then  noted  that  the 
elimination  of  urea  is  much  diminished.  In  health,  however,  this 
does  not  occur,  and  on  examination  of  the  different  tissues  and 
organs  of  the  body  such  acids  are  found  only  in  traces.  We  must 
hence  assume  that  these  acids,  supposing  them  to  occur  as  primary 
products  of  albuminous  decomposition  within  the  body,  are  trans- 
formed at  once  into  other  substances,  which  in  turn  give  rise  to  urea. 
As  all  these  bodies  on  oxidation  yield  ammonium  carbonate,  this 
substance  would  hence  suggest  itself  as  a  probable  antecedent  of 
urea.  We  find,  as  a  matter  of  fact,  that  ammonium  carbonate 
when  ingested  by  the  mouth,  or  otherwise  introduced  into  the  body, 


THE  ORGANIC  CONSTITUENTS   OF  THE    URINE.  223 

appears  in  the  urine  as  urea.     This  transformation  of  mono-arnido- 
acids  into  urea  may  be  represented  by  the  following  equations : 

(1)  CH2(NH,).COOH  +  20  =  NH4.COOH  +  C02 

Glycocoll.  Ammonium 

formate. 

(2)  2NH4.COOH  +  20  =  (NH4)2.C03  +  H20  +  CO, 

VIT 

(3)  (XH4)2.C03  =  CO<^     '     +  2H20 

\NH2 
Urea. 

Drechsel  has  further  shown  that  the  amido-acids  yield  carbamic 
acid  on  oxidation,  and  that  through  alternate  oxidation  and  reduction 
urea  can  result  from  the  ammonium  salt,  as  shown  in  the  equations  : 

NH2  NH2 

(1)  CO'  +  O    =  C0<  +  H20 

X).NH4  X).NH2 

Ammonium  Carbamic  acid. 

carbamate. 

/NH,  .NH2 

(2)  C0<  +  2H  =  C0<  +  H20 

xO.XH,  \NH2 

Carbamic  acid.  Urea. 

That  carbamic  acid  is  present  in  the  normal  acid  urine  of  man 
and  the  dog  has  been  proved.  Nencki  and  Halin,  moreover, 
observed  that  in  dogs  in  which  the  liver  was  temporarily  excluded 
from  the  general  circulation  larger  amounts  of  carbamic  acid 
appeared  in  the  urine  than  under  normal  conditions,  and  that  the 
animals  showed  symptoms  of  intoxication  identical  with  those 
observed  when  carbamates  are  directly  introduced  into  the  blood- 
current.  These  symptoms  were  also  present  when  carbamates  were 
introduced  into  the  stomach,  in  which  case  normal  dogs  show  no 
signs  of  poisoning. 

While  it  has  been  assumed  above,  that  urea  is  largely  referable  to 
a  transformation  of  mono-amido-acids  into  ammonium  carbonate  or 
carbamate,  as  the  case  may  be,  and  while  it  has  been  shown  that 
such  a  transformation  actually  does  occur,  we  must  yet  remember 
tli.it  only  traces  of  amido-acids  are  normally  found  in  the  tissues. 
There  is  reason  to  believe  that  the  greater  portion  of  the  albuminous 
nitrogen  is  normally  sel  free  from  the  various  organs  of  the  body  in 
the  form  of  the  ammonium  salt  of  paralactic  acid,  and  there  is  a 
tendency  among  physiologists  at  the  present  time  to  regard  this 
-alt  a-  the-  common  antecedent  of  urea.  It  has  been  demonstrated, 
a-  a  matter  of  fact,  that  urea  results  when  ammonium  lactate  is 
passed  through  the  isolated  liver  of  a  dog;  and  clinically,  also,  we 
observe  that  both  ammonia  and  lactic  acid  appear  in  the  urine  in 
increased  amounts  when    the    liver   is  extensively  diseased.      Similar 

results  are  obtained  in  birds,  in  which  uric  acid  represents  the 
principal  end-producl  of  nitrogenous  metabolism.  In  <^'v^v  it  is 
thus  noted  thai  after  extirpation  of  the  liver  the  greater  portion  of 
the  urinary  nitrogen  appears  in  the  form  of  ammonium  lactate. 


224  THE   URINE. 

Under  normal  conditions  it  is  assumed  that  the  lactate  is  trans- 
formed into  ammonium  carbonate,  which  in  turn  yields  the  carba- 
mate, and  the  urea  finally  results  through  a  synthetic  process,  which 
is  probably  effected  through  the  agency  of  a  certain  ferment.  We 
may  further  imagine  that  the  paralactic  acid  in  the  last  instance  may 
result  from  a  decomposition  of  the  mono-amido-radicles  of  the  albu- 
minous molecules,  and  in  this  form  the  theory  would  embrace  the 
two  outlined  above.  The  various  changes  may  be  represented  by 
the  equations  : 

(1)  2NH4.C3H503  +  120  =  (NH4)2C03  +  5C02  +  5H20 
Ammonium  lactate. 

jra2 

(2)  (KE4)2C03  =  C0<  +   H20 

X).NH4 

Ammonium 
carbamate. 

,NH2  NH2 

(3)  C0(  =  C0(  +   H20 

XO.NH4  XNH2 

Urea. 

While  we  have  seen  that  urea  may  originate  in  the  animal  body 
through  a  process  of  oxidation  only,  as  also  synthetically  through 
alternate  reductions  and  oxidations,  there  is  still  another  possibility, 
viz.,  that  it  may  be  derived  from  the  albumins  by  hydrolysis  only. 
We  know,  as  a  matter  of  fact,  that  a  number  of  nitrogenous  substances 
are  found  in  the  body,  such  as  kreatin,  kreatinin,  oxaluric  acid,  and 
others,  which  on  hydrolytic  decomposition  give  rise  to  the  formation 
of  urea,  and  it  is  quite  possible  that  a  certain  proportion  may  be 
referable  to  this  source. 

We  have  also  seen  that  on  hydrolytic  decomposition  all  albumins 
which  have  been  examined  in  this  direction  yield  comparatively 
large  amounts  of  arginin,  and  that  this  can  be  further  decomposed 
in  the  same  manner  into  ornithin  and  guanidin,  which  latter  then 
yields  urea.  As  arginin  is  now  known  to  occur  in  the  animal  body 
as  such,  there  is  no  reason  for  supposing  that  a  certain  fraction  of 
the  urea  may  not  be  formed  as  just  indicated,  and  Drechsel  indeed 
has  expressed  the  opinion  that  10  per  cent,  of  the  total  amount  may 
thus  result  through  hydrolytic  processes  only. 

Hoppe-Seyler  has  suggested  that  in  the  transformation  of  the 
mono-amido  acids  into  urea  cyanic  acid  may  be  produced  as  an  inter- 
mediary product,  and  that  urea  then  results  through  the  interaction 
of  two  molecules  of  this  acid,  as  is  shown  in  the  equation  : 

/NH2 
CONH  +  CONH   +  H20  =   CO<  +   C02. 

XNH2 

In  all  probability  a  certain  amount  of  urea  is  produced  in  the 
animal  body  in  different  ways,  and  there  is  reason  to  believe,  more- 
over, that  its  formation  is  not  confined  to  one  single  organ.  The 
greater  portion,  no  doubt,  is  formed  synthetically  in  the  liver.     Of 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  225 

this,  indeed,  we  have  abundant  proof.  It  has  been  shown  that  in 
diseases  of  this  organ  which  are  associated  with  an  extensive  destruc- 
tion of  the  glandular  elements  a  diminished  amount  of  urea  is  found 
in  the  urine,  while  ammonia  and  lactic  acid  are  present  in  increased 
quantity,  and  the  mono-amido  acids  may  appear  as  such.  In  cases 
of  this  kind  as  much  as  37  per  cent,  of  the  total  amount  of  urinary 
nitrogen  has  been  found  in  the  form  of  ammonia.  In  the  mammal, 
moreover,  symptoms  of  carbamic  acid  poisoning  are  observed  when 
the  liver  is  excluded  from  the  general  circulation,  and,  as  has  been 
shown,  the  formation  of  urea  from  ammonium  lactate  or  carbonate 
may  be  demonstrated  in  the  isolated  livers  of  dogs.  As  negative 
results  were  obtained  by  von  Schroeder  when  blood  containing  am- 
monium carbonate  was  passed  through  the  kidneys  and  through  the 
isolated  hind-quarters  of  dogs,  the  conclusion  suggests  itself  that  in 
these  organs  a  formation  of  urea  does  not  occur/  This  inference  is, 
however,  not  admissible  in  the  light  of  our  modern  knowledge  of 
the  origin  of  urea,  for  Ave  can  readily  conceive  that  although  a  syn- 
thetic formation  from  ammonium  carbonate  may  not  occur  in  these 
organs,  it  is  nevertheless  possible  that  it  may  originate  in  a  different 
manner. 

The  transfusion  experiment,  after  all,  only  shows  whether  or  not  a 
new  body  can  be  formed  in  the  organ  under  investigation  from  other 
substances  which  are  passed  through  it  as  such.  Before  deciding 
that  urea  cannot  be  produced  in  these  parts  it  would  hence  be  neces- 
sity to  experiment  with  all  those  substances  which  can  be  made  to 
yield  urea  in  the  test-tube,  and  which  we  know  to  occur  in  the  ani- 
mal body.  In  the  spleen,  where  arginin,  for  example,  is  known  to 
occur,  it  would  be  of  interest  to  ascertain  whether  urea  is  produced 
here  when  blood  containing  arginin  is  passed  through  the  organ. 

If  we  accept  the  modern  doctrine  that  urea  not  only  originates  in 
the  animal  body  in  different  ways,  but  that  it  may  also  be  formed  in 
other  organs  besides  the  liver,  we  can  also  understand  why  it  is  that 
in  certain  diseases  of  the  liver  the  diminution  in  the  formation  of 
area  is  not  always  proportionate  to  the  extent  of  parenchymatous 
degeneration,  and  that  no  case  has  been  reported  in  which  the 
formation  of  urea  ceased  altogether.  It  is  also  made  clear  why 
una  is  found  in  the  urine  of  birds  and  reptiles,  although  a  synthetic 
production  of  the  substance  manifestly  does  not  occur  in  these 
animals.  Its  origin  is  here  no  doubt  to  be  sought  in  its  formation 
from  -uch  bodies  as  kreatin,  kreatinin,  arginin,  and  the  like. 

Nitrogenous   Equilibrium. — The  albumins,  of  course,  are   the  ulti- 
mate source  of  the  urea.     According  to    Pettenkofer,  they  exist  in 
the  body  in  two  forms,  viz.,  :i^  organized  albumin  which  is  built  up 
into  tissues,  and  n-  so-called  circulating  albumin  which  is  present  in 
js  of  what   i-  actually  required,  and  is  broken  down  directly 

and  eliminated  in  the  urine  without  having  entered  into  the  con- 
struction of  the  body  proper.  This  portion  of  the  albumin  furnishes 
the  greater  portion  of  the  urea,  while  the  organized  albumin  repre- 

15 


226  THE   URINE. 

sents  a  minor  but  more  constant  source.     The  actual  amount  that  is 
eliminated  is  thus  primarily  dependent  upon  the  amount  ingested. 

The  total  urinary  nitrogen  is  under  normal  conditions  practically 
equivalent  to  the  quantity  ingested,  barring  the  small  fraction  which 
escapes  digestion  in  the  feces.  Such  a  condition  is  spoken  of  as  the 
nitrogenous  equilibrium  of  the  body.  Of  this,  however,  different 
levels  may  exist,  which  may  vary  in  the  same  individual.  If  the 
amount  of  nitrogenous  food  is  thus  diminished,  the  amount  of 
urinary  nitrogen  will  also  decrease ;  and  if  the  amount  of  food  then 
remains  constant,  the  nitrogenous  output  will  likewise  remain  the 
same.  If,  on  the  other  hand,  more  nitrogen  is  now  ingested,  an 
increased  elimination  will  result ;  but  a  certain  fraction  is  retained 
by  the  body,  and  gradually  a  higher  level  of  equilibrium  becomes 
established. 

There  are  natural  limits  to  this  power  of  accommodation,  how- 
ever, and  we  finally  reach  a  point  which  varies  in  different  indi- 
viduals, where  a  further  increase  in  the  amount  of  nitrogen  that  is 
ingested  does  not  lead  to  a  higher  level  of  equilibrium,  and  where 
consequently  a  further  retention  of  nitrogen  does  not  occur.  Over- 
feeding then  results  in  various  digestive  disturbances — diarrhoea  and 
vomiting  occur,  and  the  body. thus  protects  itself  against  an  undue 
accumulation  of  circulating  albumin  which  it  would  not  be  able  to 
dispose  of  in  a  normal  manner. 

Underfeeding,  on  the  other  hand,  gradually  leads  to  an  increased 
destruction  of  the  organized  albumins.  For  a  while  the  reserve  of 
fats  and  carbohydrates  is  still  capable  of  protecting  the  body  against 
an  unduly  rapid  loss  of  nitrogen  from  this  source,  but  death  finally 
results. 

From  the  fact  that  the  level  of  nitrogenous  equilibrium  is  different 
in  different  people  and  may  vary  in  one  and  the  same  individual,  it 
follows  that  the  amount  of  urea  also  must  vary.  Any  figures  indi- 
cating the  amount  of  urea  that  is  eliminated  in  the  urine  can  there- 
fore be  of  little  value  unless  we  are  acquainted  with  the  actual  state 
of  health  of  the  individual,  his  body-weight,  his  habits  of  life  as 
regards  exercise,  the  amount  of  nitrogenous  food  ingested,  etc. 
Having  a  knowledge  of  all  these  factors,  however,  we  may  be  able 
to  sav  whether  the  "amount  of  urea  is  normal  or  not.  Certain  figures 
have'been  given  by  physiologists  to  indicate  the  amount  of  nitrogen- 
ous food  which  should  enter  into  the  composition  of  the  diet,  and 
from  these  we  can  approximately  calculate  the  amount  of  urea  that 
should  appear  in  the  urine.  By  estimating  this  in  turn,  or  still 
better,  of  course,  the  total  amount  of  nitrogen,  we  can  accordingly 
decide  whether  or  not  the  individual  is  consuming  a  sufficient  amount 
of  nitrogen  in  his  food.  The  figures,  however,  have  been  constructed 
withoutdue  regard  to  the  factors  above  indicated,  and  are  in  my 
opinion,  at  least,  too  high  as  averages. 

I  am  willing  to  admit  that  an  elimination  of  40-50  grammes  of 
urea  may  be  normal  in  certain  cases,  as  in  soldiers  on  forced  marches, 


THE  ORG  A  SIC  CONSTITUENTS  OF  THE   URINE.  'I'll 

among  the  laboring-classes,  etc.,  but  I  should  certainly  look  upon 
the  average  merchant  or  student,  who  leads  a  sedentary  life,  as  an 
overfed  individual  if  his  daily  elimination  of  urea  should  exceed  30 
grammes  in  the  twenty-four  hours.  Among  the  well-to-do  classes 
I  find  that  an  elimination  of  from  20  to  25  grammes  is  probably 
normal,  taking  the  body-weight  of  the  person  into  due  considera- 
tion. A  smaller  amount  even  is  not  infrequently  met  with  in  people 
of  sedentary  habits  who  are  in  perfect  health,  but  I  should  scarcely 
regard  such  a  quantity  as  normal  for  the  average  laboring-man. 
While  extensive  variations  in  the  amount  of  urea  are  thus  observed 
in  health,  still  greater  deviations  from  average  figures  are  noted  in 
disease,  but  here  as  there  we  must  always  take  into  account  the 
amount  of  nitrogen  that  is  ingested  and  the  body  weight. 

An  increase  in  the  elimination  of  urea,  referable  to  the  destruction 
of  organized  albumins,  is  here  frequently  observed,  but  may  be 
obscured,  owing  to  a  deficient  ingestion  of  nitrogen,  unless  the  amount 
of  the  latter  is  known.  At  this  place,  however,  it  is  scarcely  neces- 
sary to  enter  into  pathological  considerations,  and  I  must  refer  the 
reader  to  other  works  for  detailed  information  on  such  questions. 
But  I  may  briefly  recall  that  in  certain  diseases  of  the  liver  in 
which  an  extensive  destruction  of  the  parenchyma  is  taking  place 
the  amount  of  urea  may  be  greatly  diminished,  although  a  fairly 
abundant  supply  of  nitrogenous  food  is  ingested.  As  has  been 
shown,  the  synthetic  formation  of  urea  is  here  seriously  impeded, 
and  as  a  result  we  find  that  a  considerable  proportion  of  the  urinary 
nitrogen  then  appears  in  the  form  of  ammonium  salts  of  paralactic 
acid,  of  carbamic  acid  and  carbonic  acid,  and  in  extreme  cases, 
indeed,  mono-amido  acids,  such  as  leucin  and  tyrosin,  may  be  found. 

Properties  of  Urea. — Urea  crystallizes  in  two  forms,  viz.,  in 
loiv/,  i'uiQ  white  needles  if  rapidly  formed,  or  in  long,  colorless 
rhombic  prisms  when  allowed  to  crystallize  more  slowly  from  its 
solutions. 

It  melts  at  130°  to  132°  C,  but  is  probably  decomposed  already 

at  a  temperature  of  100°  C.     It  is  readily  soluble  in  water  and 

alcohol,  but  is  insoluble  in  anhydrous  ether,  chloroform,  and  benzol. 

A-  the  substance  is  an  acid  amide,  its  solutions  present  a  neutral 

ion. 

In  accordance  with  its  character  as  an  unsaturated  amide  of 
carbonic  acid,  however,  it  combines  with  acids  to  form  crystalline, 
salt-like  compounds.  The  most  important  of  these  are  the  nitrate 
and  the  oxalate. 

Urea  nitrate,  ( K )( X  I  L)_,. II  .V  )..,  crystallizes  in  two  forms,  viz.,  in 
delicate  rhombic,  horizontal  platelet-,  which  :ire  commonly  arranged 
overlapping  in  a  shingle-like  manner  when  rapidly  formed,  or  as 
thicker  rhombic  columns  or  plate-  when  allowed  to  crystallize  more 
.-lowly. 

Qrea  nitrate  is  readily  soluble  in  distilled  water,  but  dissolves 
with   difficulty  if  this    is  acidulated    with   nitric  acid,  and  also  in 


228  THE   URINE. 

alcohol.  Its  formation  is  frequently  observed  when  urine  contain- 
ing much  urea  is  examined  tor  albumin  in  the  cold  with  nitric 
acid.  On  standing,  the  nitrate  may  then  separate  out  in  crystalline 
form.  On  heating,  the  substance  is  decomposed  without  leaving  a 
residue. 

Urea  oxalate,  CO(NH2)2.C2H204,  crystallizes  in  rhombic  plates,  or 
hexagonal  prisms,  and  is  less  soluble  in  water  than  the  nitrate ;  in 
alcohol  and  in  dilute  solutions  of  oxalic  acid  it  is  nearly  insoluble. 
The  substance  is  obtained  in  crystalline  form  on  adding  a  saturated 
solution  of  oxalic  acid  to  a  concentrated  solution  of  urea. 

Urea  also  combines  with  various  neutral  salts,  such  as  sodium 
chloride  and  ammonium  chloride,  and  also  with  the  nitrates  of 
sodium  and  the  oxides  of  silver  and  mercury,  to  form  double  salts. 
With  mercuric  nitrate  three  different  compounds  result,  according 
to  the  concentration  of  the  two  solutions,  viz.,  CO(NH2)2.Hg2(N03)4, 
CO(NH2)2.Hg3(N03)6,  and  .  [CO(NH2)2]2.Hg(N03)2  +  3HgO.  The 
latter  compound  is  of  special  interest,  as  Liebig's  quantitative  esti- 
mation of  urea,  which  was  formerly  much  employed,  was  based 
upon  its  formation.  It  results  when  a  2  per  cent,  solution  of  urea 
is  treated  with  a  feebly  acid  solution  of  mercuric  nitrate,  and  the 
mixture  is  subsequently  neutralized. 

Mercuric  chloride  precipitates  urea  in  alkaline,  but  not  in  neutral 
solutions. 

Very  important,  further,  is  the  behavior  of  urea  toward  sodium 
hypochlorite  or  hypobromite,  as  the  most  usual  method  of  esti- 
mating urea  in  the  clinical  laboratory  is  based  upon  the  reaction 
which  here  takes  place.  This  reaction  may  be  represented  by  the 
equation  : 

CO(NH2)2  +  SNaOBr  =  3NaBr  +  C02  +  2H20  +  2N. 

In  practical  work  an  alkaline  solution  of  the  hypobromite  is 
employed,  so  that  the  carbon  dioxide  which  is  liberated  is  at  once 
absorbed,  while  the  nitrogen  remains.  It  is  to  be  noted,  however, 
that  while  1  gramme  of  urea  should  theoretically  give  rise  to  the 
formation  of  372.7  c.c.  of  nitrogen,  354.3  c.c.  are  at  best  obtained 
at  0°  C.  and  a  pressure  of  760  Hgmm.  In  clinical  work  this 
difference  is  unimportant,  and  it  is  in  a  measure  equalized  by  the 
evolution  of  a  small  amount  of  nitrogen  from  some  of  the  other 
nitrogenous  constituents  which  are  at  the  same  time  present.  On 
hydrolysis  urea  is  transformed  into   ammonium   carbonate  : 

CO(NH2)2  +  2H20  =  (NH4)2C03. 

This  occurs  during  the  process  of  ammoniacal  fermentation,  which 
results  when  urine  is  exposed  to  the  air,  and  is  referable,  as  I  have 
pointed  out,  to  the  action  of  a  specific  bacterial  enzyme.  On  boiling 
with  acids  or  alkalies  the  same  result  is  primarily  obtained,  but  the 
salt  is  then  further  decomposed,  with  the  liberation  of  carbon  dioxide 


THE  ORGANIC  CONSTITUENTS   OF  THE   URINE.  229 

and  ammonia,  respectively.  This  decomposition  is,  however,  also 
noted  during  the  process  of  ammoniacal  fermentation. 

On  heating  the  substance  in  aqueous  solution  in  a  sealed  tube  to 
a  temperature  of  100°  C.  ammonia  and  carbon  dioxide  likewise 
result. 

Nitrous  acid  when  added  in  excess  decomposes  urea,  with  the 
formation  of  nitrogen,  carbon  dioxide,  and  water,  but  the  acid  is  at 
the  same  time  decomposed,  as  is  seen  in  the  equation  : 

CO(NH2)2  +  2HN02  =  C02  +  4N  +  3H20. 

This  reaction  is  utilized  when  it  is  desired  to  remove  nitrous  acid 
from  a  solution. 

On  heating  the  dry  substance  in  a  test-tube  to  a  temperature  of 
about  150°-i70°  C,  fumes  of  ammonia  are  freely  evolved,  owing  to 
the  decomposition  of  the  urea  with  the  formation  of  biuret,  as  shown 
in  the  equation  : 

/NH2 
CCK 

2CO(NH2)2  =  NH3  +       NNH 

XNH2 
Biuret. 

On  further  heating,  more  ammonia  is  given  off;  the  melted  mass 
finally  solidifies,  and  may  be  shown  to  contain  both  biuret  and 
cyanuric  acid.  The  reaction  which  takes  place  may  be  represented 
as  follows  : 

(1)  3CO(NH2)2   ==  3CONH  +  3NH3 
Cyanic  acid. 

(2)  3CONH  =  C3N3(OH)3 

Cyanuric  acid. 

To  demonstrate  the  presence  of  the  biuret,  the  residue  is  dis- 
solved in  a  dilute  solution  of  sodium  hydrate,  when  upon  the  care- 
ful addition  of  a  dilute  solution  of  copper  sulphate  a  beautiful, 
purple-red  color  develops  (see  also  page  34). 

A  very  delicate  test  also  is  the  following  :  2  c.c.  of  a  concentrated 
solution  of  furfurol  arc-  treated  with  4-b'  drops  of  strong  hydro- 
chloric acid.  If  to  this  mixture,  which  should  not  present  a  red 
color,  a  small  crystal  of  urea  is  then  added,  a  deep  violet  develops 
in  the  course  of  a  few  minutes. 

Synthetic  Formation. — As  has  been  mentioned,  urea  was  the  first 
organic  substance  formed  in  the  animal  body  which  was  made  syn- 
thetically in  the  chemical  laboratory.  Wohler  in  1828  produced  the 
substance  artificially  by  heating  ammonium  cyanate  to  a  temperature 
of  loo-  C.,  when  a  rearrangemenl  of  atoms  occurs  and  urea  results : 

MI, 
(NH4)CN0       CO 

Other  methods  now  exisl    by  which  urea    can    also    be    made   syn- 


230     '  THE    URINE. 

thetically,  but  they  are  all  more  or  less  modifications  of  the  one  just 
described. 

Isolation  from  the  Urine. — To  isolate  urea  on  a  small  scale,  50-100 
c.c.  of  urine  are  evaporated  to  a  syrup  on  a  water-bath  and  ex- 
tracted with  150  c.c.  of  strong  alcohol  by  rubbing  in  a  mortar. 
The  alcoholic  extract  is  filtered,  the  alcohol  distilled  off,  and  the 
syrupy  residue  treated  with  concentrated  nitric  acid  in  the  cold. 
The  urea  nitrate  which  crystallizes  out  on  standing  is  filtered 
off  with  a  suction-pump,  dissolved  in  hot  water,  and  the  aqueous 
solution  decolorized  by  gently  heating  with  animal  charcoal.  The 
colorless  filtrate  is  then  treated  with  barium  carbonate  in  substance 
so  long  as  carbon  dioxide  is  evolved,  and  finally  rendered  alkaline 
with  barium  hydrate  solution.  The  urea  is  thus  liberated  according 
to  the  equation  : 

2CO(NH2)2.HN03  +  BaC03  =  Ba(N03)2  +  2CO(NH2)2  +  H20  +    C02. 

The  solution  is  now  evaporated  to  dryness  and  the  residue  extracted 
with  absolute  alcohol.  On  concentrating  this  extract  the  urea  crys- 
tallizes out  in  colorless  prisms,  which  may  then  be  treated  as  above 
indicated. 

Quantitative  Estimation. — In  the  clinical  laboratory  the  old  method 
of  Knop  and  Hufner  is  almost  exclusively  employed.  This  is  based 
upon  the  decomposition  of  urea  with  sodium  hypobromite  in  alkaline 
solution,  as  already  described.  The  nitrogen  which  is  thus  liber- 
ated is  measured  and  the  corresponding  amount  of  urea  determined 
by  calculation. 

For  scientific  purposes,  however,  this  method  in  any  one  of  its 
numerous  modifications  is  not  sufficiently  accurate,  as  the  actual 
volume  of  nitrogen  which  is  obtained  always  falls  somewhat  short 
of  the  theoretical  amount.  The  sodium  hypobromite,  moreover, 
also  causes  a  partial  decomposition  of  other  nitrogenous  constituents 
of  the  urine,  and  as  the  resulting  amount  of  nitrogen  is  not  con- 
stant, a  further  error  is  incurred.  In  scientific  research  we  are 
hence  forced  to  resort  to  some  other  procedure,  such  as  that 
proposed  by  Morner  and  Sjoquist,  or  the  simpler  method  which  has 
recently  been  suggested  by  Folin. 

Method  of  Morner  and  Sjoquist. — This  is  based  upon  the 
fact  that  the  organic  nitrogenous  constituents  of  the  urine,  with  the 
exception  of  urea  and  ammonia,  are  precipitated  by  an  alkaline 
barium  chloride  mixture.  This  precipitate  is  insoluble  in  ether- 
alcohol,  while  urea  and  ammoniacal  salts  are  dissolved  together  with 
a  small  amount  of  barium  hydrate.  In  the  concentrated  ether- 
alcoholic  filtrate  the  nitrogen  is  then  determined  according  to  Kjel- 
dahl's  method,  after  the  ammoniacal  salts  have  been  decomposed, 
and  the  resulting  ammonia  has  been  driven  on0.  From  the  per- 
centage of  nitrogen  the  corresponding  amount  of  urea  is  then  cal- 
culated by  multiplying  by  2.14. 

Five   c.c.  of  urine  are  treated  with  an  equal  volume  of  baryta 


THE   ORGANIC  CONSTITUENTS  OF  THE   URINE.  231 

mixture,  which  contains  250  grammes  of  barium  chloride  in  1000 
c.c.  of  a  5  per  cent,  solution  of  barium  hydrate.  To  this  are  added 
100  c.c.  of  a  mixture  of  two  parts  of  97'  per  cent,  alcohol  and  one 
part  of  ether.  After  twenty-four  hours  the  precipitate  is  filtered 
off  and  treated  with  100  c.c.  of  ether-alcohol.  Filtrate  and  wash- 
ings are  then  concentrated  at  a  temperature  not  exceeding  60°  C. 
to  about  20  c.c,  or  to  a  point  where  ammonia  is  no  longer  evolved, 
which  can  be  recognized  by  testing  the  vapor  with  litmus-paper. 
A  small  amount  of  water  may  be  added  if  the  solution  should 
become  too  concentrated.  It  is  then  washed  into  a  Kjeldahl  digest- 
ing flask  with  as  little  water  as  possible,  to  which  a  few  drops  of 
concentrated  sulphuric  acid  have  been  added.  The  solution  is  now 
concentrated  to  a  very  small  volume,  upon  a  water-bath,  and  treated 
with  20  c.c.  of  a  mixture  of  two  parts  of  concentrated  sulphuric 
acid  and  one  part  of  the  fuming  acid.  A  pinch  of  yellow  oxide  of 
mercury  (about  0.3  gramme)  is  further  added,  when  the  process  is 
continued  according  to  Kjeldahl,  as  described  below. 

Method  of  Folix. — This  is  based  upon  the  following  considera- 
tions :  crystallized  magnesium  chloride,  MgCl2.6H20  boils  in  its 
water  of  crystallization  at  a  temperature  of  about  160°  C.  Urea 
is  quantitatively  decomposed  in  such  a  solution  into  ammonia  and 
carbon  dioxide  within  one-half  hour.  If  the  process  is  carried  out 
in  acid  solution,  the  ammonia  can  subsequently  be  distilled  off  after 
rendering  the  mixture  alkaline,  and  is  then  titrated.  The  cor- 
responding amount  of  urea  is  ascertained  by  calculation.  At  the 
same  time,  however,  the  preformed  ammonia  is  obtained,  and  it  is 
hence  necessary  to  eliminate  this  source  of  error  by  a  separate 
estimation  of  this  form.  This  is  conveniently  done  "according  to 
the  method  which  has  likewise  been  suggested  by  Folin  (sec  below). 

Method. — Three  c.c.  of  urine  are  placed  in  an  Erlenmeyer  flask 
of  200  c.c.  capacity,  together  with  20  grammes  of  magnesium 
chloride  and  2  c.c.  of  concentrated  hydrochloric  acid.  (The  magne- 
sium chloride  usually  contains  a  small  amount  of  ammonia,  which 
must  be  separately  determined.)  The  flask  is  closed  with  a  per- 
forated stopper  through  which  a  straight  glass  tube  passes,  measur- 
ing 200  mm.  in  length,  with  a  diameter  of  10  nun.  The  mixture  is 
now  boiled  until  the  drops  flowing  back  through  the  tube  produce 
a  hissing  sound  on  coming  in  contact  with  the  solution.  Alter  this 
point  has  been  reached,  the  boiling  is  continued  more  moderately  for 
twenty-five  to  thirty  minutes.  The  solution  while  still  hot  is  care- 
fully diluted  to  about  500c.C. — at  first  by  allowing  the  water  to  How 
drop  by  drop  through  the  tube;    it   is  then  transferred  to  a   ]()()()  c.c. 

retort,  treated    with    about  7  or  8  <•.<■.  of*  a   20  per  cent,  solnti. f 

-odium  hydrate,  and  the  ammonia  distilled  oil'  into  a  measured 
amount,  of  a  decinormal  solution  of  sulphuric  acid.      The  distillation 

may  be  interrupted  when  about  350  c.c.  have  passed  over  (viz.,  after 
about  sixty  minutes).  The  distillate  is  boned  lor  ,-i  moment  to 
remove  any  carbon   dioxide   which   may  he  presenl   in  solution,  and 


232  THE    URINE. 

on  cooling  is  titrated  to  determine  the  excess  of  acid.  Each  cubic 
centimeter  of  the  decinormal  ammonia  present  in  the  distillate  cor- 
responds to  0.003  gramme,  viz.,  to  0.1  per  cent  of  urea. 

From  this  result  the  amount  of  preformed  ammonia  and  that 
present  in  the  20  grammes  of  magnesium  chloride  must  be  deducted. 

If  desired,  the  estimation  can  also  be  made  with  the  urea-con- 
taining nitrate  obtained  with  Morner  and  Sjoquist's  method,  but 
Folin  states  that  the  previous  isolation  of  the  urea  in  such  manner 
is  probably  not  necessary. 

Estimation  of  the  Preformed  Ammonia  (according  to  Folin). — 
Ten  c.c.  of  urine  are  diluted  to  about  450  c.c,  treated  with 
a  small  amount  of  burnt  magnesia  (0.5  gramme)  and  boiled  for 
forty-five  minutes,  the  distillate  being  received  in  decinormal  sul- 
phuric acid.  The  ammonia  is  then  determined  by  titration  as 
above.  As  a  small  amount  of  urea,  however,  is  decomposed  during 
the  prolonged  ebullition,  it  is  necessary  to  ascertain  separately  the 
quantity  of  ammonia  which  is  referable  to  this  source.  To  this  end, 
the  retort  is  opened  at  the  expiration  of  forty-five  minutes,  and  an 
amount  of  water  added  which  is  approximately  equivalent  to  that 
of  the  distillate.  The  distillation  is  then  continued  for  another 
period  of  forty-five  minutes,  the  distillate  received  in  decinormal 
sulphuric  acid,  and  the  ammonia  referable  to  decomposition  of  the 
urea  estimated  as  before.  The  difference  between  the  two  results 
indicates  the  amount  of  preformed  ammonia  that  was  originally 
present. 

Estimation  of  the  Total  Urinary  Nitrogen. — Kjeldahl's  Method. 
— The  method  is  based  upon  the  observation  that  on  treating  urine 
with  a  mixture  of  two  parts  of  concentrated  sulphuric  acid  and  one 
part  of  fuming  sulphuric  acid  and  boiling,  the  entire  amount  of 
nitrogen  can  be  transformed  into  ammonium  sulphate.  This  is 
then  decomposed  with  an  excess  of  sodium  hydrate  and  the  liberated 
ammonia  estimated  by  distilling  into  a  known  amount  of  dilute  acid, 
and  retitrating  the  excess  of  acid. 

Five  c.c.  of  urine  are  treated  in  a  Kjeldahl  digesting  flask  with 
a  pinch  of  yellow  oxide  of  mercury  (about  0.3  gramme)  and  20  c.c. 
of  the  sulphuric  acid  solution.  The  mixture  is  boiled  until  a  per- 
fectly colorless  solution  is  obtained.  Vigorous  ebullition,  how- 
ever, must  be  avoided,  and  the  flask  should  be  placed  at  an  angle 
of  about  45  degrees,  so  as  to  prevent  loss  from  spurting.  The  milder 
the  ebullition  the  better.  On  cooling,  the  contents  of  the  flask  are 
transferred  to  a  Kjeldahl  retort,  with  the  aid  of  a  little  distilled  water. 
Sodium  hydrate  solution  (27  per  cent.)  is  then  added  until  the  greater 
portion  of  the  acid  is  neutralized.  The  fluid  is  allowed  to  cool  again, 
and  a  few  pieces  of  granulated  zinc  or  a  little  talcum  is  thrown  in, 
when  a  mixture  of  the  hydrate  solution  and  of  a  4  per  cent,  solution 
of  potassium  sulphide  is  further  added  in  excess.  Of  either  solution, 
40  c.c.  are  added  in  all.  The  addition  of  the  latter  is  necessary,  as 
the  mixture  not  only  contains  ammonium  sulphate,  but  also  amido- 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  233 

■compounds  of  mercury,  which  latter  would  not  give  up  their  entire 
amount  of  nitrogen  if  sodium  hydrate  alone  were  present.  The  talcum 
or  zinc  merely  prevents  an  unduly  violent  bumping  when  boiling. 
The  retort  is  then  immediately  connected  with  a  condenser  through 
the  intervention  of  a  Kjeldahl  distilling  tube.  The  condensing  tube 
dips  into  a  nitrogen  bulb,  which  contains  a  carefully  measured 
amount  of  a  one-fourth  normal  solution  of  sulphuric  aeid  ;  30  c.c. 
are  usually  sufficient.  The  mixture  is  now  distilled  until  about 
two-thirds  have  passed  over.  The  condenser  is  rinsed  with  a 
little  distilled  water,  which  is  added  to  the  distillate.  After  the 
addition  of  a  few  drops  of  tincture  of  cochineal  the  excess  of 
acid  is  retitrated  with  a  one-fourth  normal  solution  of  sodium 
hydrate.  The  difference  indicates  the  amount  of  acid  which  was 
consumed  in  uniting  with  the  liberated  ammonia.  As  1  c.c.  of  the 
one-fourth  normal  solution  represents  0.0035  gramme  of  nitrogen, 
the  amount  contained  in  the  5  c.c.  of  urine  is  ascertained  by  multi- 
plying the  number  of  cubic  centimeters  employed  by  this  figure, 
from  which  the  total  amount  of  twenty-four  hours  is  then  readily 
calculated.  The  corresponding  amount  of  albumin  is  obtained  by 
multiplying  this  figure  by  6.25.  The  method,  as  just  described, 
appears  simple  enough,  but  in  reality  requires  a  considerable  amount 
of  experience  to  obtain  figures  that  are  reliable.  With  experience, 
however,  the  method  is  exceedingly  accurate.  In  every  case,  of 
course,  chemically  pure  reagents  are  necessary,  and  it  is  well  to  test 
these  with  care  before  proceeding. 

Uric  Acid. 

Whereas  in  mammals,  the  amphibia,  and  fishes  urea  is  the  most 
important  end-product  of  nitrogenous  metabolism,  the  greater  por- 
tion of  the  urinary  nitrogen  in  birds  and  reptiles  is  eliminated  as 
uric  aeid. 

Origin. — The  formation  of  uric  acid  in  birds  and  reptiles  is  analo- 
gous t<>  the  formation  of  urea  in  the  mammal.  It  is  derived  in 
thi'  last  instance  from  the  albumins  of  the  tissues  and  from  the 
ing  -I'd  food,  and,  like  urea,  is  formed  synthetically  in  the  liver. 
Tin-  i-  true  at  leasl  of  the  greater  portion  ;  while  a  variable  frac- 
tion originates  from  the  nucleins,  viz.,  the  xanthin  bases.  Organic 
ammonium  -alt-,  amido-acids,  urea,  and  ammonium  carbonate,  when 
<ri vii  to  birds  in  their  food,  appear  in  the  urine  as  uric  aeid,  and 
it  i-  now  thought  that  here  also  i he  greater  portion  of  the  nitrogen 
i-  carried  to  the  liver  n-  ammonium  lactate.  We  accordingly  find 
thai  after  extirpation  of  the  liver  almost  all  the  urinary  nitrogen 
are  in  thi-  form,  and  that  ammonium  carbonate  when  given  by 
th<-  mouth  i-  eliminated  a-  such.  Of  the  manner  in  which  the 
Byn thesis  of  uric  acid  i-  effected  in  the  liver,  however,  we  know 
but    little.       Urea    or  ammonium   carbonate  cannot,   of  course,   give 

rise  to  it-  formation  alone,  as  the  available  amount  of  carbon  i- 


23  i  THE   URINE. 

too  small.  We  must  hence  assume  that  some  other  substance  enters 
into  the  reaction.  This  substance,  however,  is  as  yet  unknown,  but 
we  may  imagine  that  one  portion  of  ammonium  lactate  is  first  trans- 
formed into  ammonium  carbonate,  and  that  the  uric  acid  is  then 
formed  through  the  union  of  this  with  another  molecule  of  lactic 
acid.  Horbaczewski,  indeed,  has  shown  that  artificially  uric  acid 
may  be  formed  from  lactic  acid,  ammonia,  and  carbon  dioxide,  by 
heating  trichloro-lactic  amide  together  with  urea.  The  reaction 
which  takes  place  may  be  represented  by  the  equation  : 

2CO(NH2)2  +  C3Ci302H2.NH2  =  NH4C1  +  2HC1  +  H20  +  C5H,N403- 
Trichloro-lactic  '  Uric  acid, 

amide. 

On  the  other  hand,  it  is  possible  that  uric  acid  may  result  through 
the  union  of  glycocoll  and  urea,  and  artificially  this  synthesis  can 
indeed  be  effected.  We  have  seen,  moreover,  that  on  hydrolytie 
decomposition  uric  acid  yields  ammonia,  carbon  dioxide,  and  gly- 
cocoll. In  this  case  the  resulting  reaction  could  be  expressed  by 
the  equation : 

3CO(NH2)2  +  CH2(NH2).COOH  =  3NH3  +  2H20  +  C5H4N403- 

While  the  greater  portion  of  the  uric  acid  is  thus  formed  syn- 
thetically in  the  liver  of  birds  and  reptiles,  a  variable  but  much 
smaller  amount  results  directly  from  the  xanthin  bases  through  a 
process  of  oxidation. 

These  are  in  part  derived  from  disintegrating  cells  of  the  body,, 
and  are  to  a  certain  extent  also  referable  to  the  ingested  food.  In 
man  and  most  mammals  this  is  indeed  the  only  source  of  the  uric 
acid,  and  through  the  researches  of  Horbaczewski  we  now  know 
that  the  nuclear  uric  acid,  as  we  may  term  it,  is  formed  together 
with  the  xanthin  bases  in  all  organs  of  the  body,  and  is  most 
abundantly  produced  in  those  which  are  especially  rich  in  nuclei, 
such  as  the  spleen  and  the  lymph-glands.  The  very  interesting 
observation  was  further  made  that  larger  amounts  of  uric  acid  could 
be  obtained  from  these  parts  when  the  blood  used  in  the  transfu- 
sion experiments  contained  much  oxygen,  while  with  venous  blood 
xanthin  bases  only  were  produced.  In  the  amphibia  and  fish,  in 
which  the  oxidation-processes  are  especially  sluggirh,  we  accordingly 
find  xanthin  bases,  but  little  or  no  uric  acid.  The  interesting  ques- 
tion now  suggests  itself,  Why  is  it  that  in  mammals  uric  acid  appears 
in  the  urine  at  all  in  view  of  the  fact  that  uric  acid  which  is  intro- 
duced into  the  stomach  is  eliminated  as  urea?  A  final  answer  to 
this  question  cannot  be  given,  but  there  is  reason  to  suppose  that  the 
uric  acid  is  here  first  carried  to  the  liver,  and  is  probably  oxidized 
to  urea  by  the  oxidizing  ferments  of  this  organ.  We  find,  as  a 
matter  of  fact,  that  an  increased  elimination  of  uric  acid  results  at 
once  when  the  blood  of  the  portal  vein  is  prevented  from  flowing 
through  the  liver  by  establishing  a  so-called  Eck  fistula  between 
this  and  the  inferior  cava,  and  when  the   hepatic  artery  is  at  the 


THE  ORG  A  NIC  CONSTITUENTS  OF  THE   URINE.  235 

same  time  ligated.  In  this  manner  the  blood  of  the  spleen  and  the 
extensive  lymphatic  districts  of*  the  intestinal  tract  is  carried  directly 
into  the  general  circulation,  and  the  combined  xanthin  bases  and 
uric  acid  hence  find  their  way  into  the  urine  without  being  sub- 
jected to  the  action  of  the  oxydases  of  the  liver.  We  may  hence 
conclude  that  the  appearance  of  these  bodies  in  the  urine  is  under 
normal  conditions,  owing  to  the  fact  that  not  all  the  blood  of  the 
bodv  reaches  the  liver  before  being  carried  to  the  kidneys. 

In  birds  and  reptiles  we  have  also  seen  that  a  certain  amount 
of  urea  appears  in  the  urine,  and,  as  I  have  already  explained,  this 
is  no  doubt  produced  directly  in  the  tissues.  As  ingested  urea  is 
here  transformed  into  uric  acid,  we  must  hence  assume  that  the 
portion  which  is  eliminated  in  the  urine  has  reached  the  kidneys 
without  having  previously  passed  through  the  liver,  and  the  process 
is  thus  quite  analogous  to  what  we  observe  in  mammals  in  the  case 
of  uric  acid. 

The  recognition  of  the  fact  that  uric  acid  in  man  has,  so  far  as 
we  know,  but  one  source,  viz.,  the  nucleins,  is  of  great  importance 
from  the  standpoint  of  pathology.  For,  whereas  normally  the  elimi- 
nation of  urea  rarely  exceeds  one  gramme  in  twenty-four  hours, 
much  larger  amounts  may  appear  in  the  urine  under  the  most 
diverse  conditions. 

In  leukaemia  especially,  a  greatly  increased  elimination  is  thus 
commonly  observed,  and  is  here  no  doubt  referable  to  the  increased 
destruction  of  leucocytes.  But  excessive  amounts  of  uric  acid 
may  also  occur  in  conditions  in  which  there  is  no  direct  evidence 
of  increased  nuclear  destruction.  In  many  cases  of  this  kind  the 
increased  elimination  is  apparently  dependent  upon  the  amount  of 
animal  food  that  is  ingested,  and  it  would  appear  that  in  such  cases 
the  liver  lias  lost  to  a  greater  or  less  degree  its  power  of  oxidizing 
the  uric  acid,  which  reaches  it  from  this  source.  Were  this  true,  we 
should  then  also  expect  that  relatively  larger  amounts  of  xanthin 
bases  should  find  their  way  into  the  urine,  and  this  indeed  may 
actually  occur.  But,  on  the  other  hand,  an  increased  elimination  of 
uric  acid  and  xanthin  bases  may  also  be  observed  although  the 
patient  has  been  plaeed  on  a  diet  which  is  practically  i'vee  from 
uuclear  nucleins.  An  adequate  explanation  of  such  an  occurence  is 
08     vet     wanting.       We    may    here   also   suppose   that     the    liver    has 

losl  it-  power  of  oxidation  so  far  as  the  alloxuric  bodies  are  con- 
cerned. Hut  we  must  bear  in  mind  that  uric  acid  is  formed  in  all 
the    tissues    of    the    body,  and    that   the   relative  amount  which  thus 

originates,  as  compared  with  the  xanthin  bases,  is  largely  influenced 
by  the  intensity  of  the  processes  of  oxidation.  It  is  hence  also 
conceivable  thai    in   such   cases  these  may  be  deficient,  while  the 

liver  may   function    in  a   normal    mi er.      The   possibility  of  a 

synthetic  production  of  uric  acid,  finally,  may  also  enter  into 
consideration. 

The  question  of  the  nature  of  the  so-called  uric  acid  diathesis  is 


236  THE   URINE. 

thus  still  in  statu  quo,  and  the  same  may  be  said  of  the  formation 
of  uratic  deposits  in  the  joints  and  tendons  in  gout.  It  appears, 
however,  that  an  increased  production  of  uric  acid,  contrary  to 
what  was  formerly  supposed,  plays  no  role  in  the  causation  of  the 
latter  disease. 

Properties  of  Uric  Acid. — Pure  uric  acid  crystallizes  in  trans- 
parent, colorless  rhombic  platelets,  the  angles  of  which  are  often 
rounded  oif.  Such  crystals  are  at  times  seen  in  urinary  sediments, 
but  more  commonly  the  substance  is  here  found  in  the  form  of 
brownish-yellow  whetstone-like  crystals,  which  may  occur  singly, 
but  are  frequently  arranged  in  groups.  These  are  quite  character- 
istic, and  cannot  be  confounded  with  crystals  of  any  other  substance 
that  may  occur  in  the  urine. 

A  great  many  other  forms  may,  however,  also  be  encountered, 
such  as  dumb-bells,  somewhat  irregular  hexagonal  platelets,  paddle- 
shaped  crystals,  etc.,  the  nature  of  which  is  not  at  once  apparent. 

Uric  acid  is  almost  insoluble  in  cold  water  (1  :  40,000),  with 
difficulty  also  in  boiling  water  (1  :  1800),  and  insoluble  in  alcohol 
and  ether. 

In  concentrated  sulphuric  acid  and  boiling  glycerin  it  dissolves 
with  comparative  ease  and  without  undergoing  decomposition.  It  is 
a  dibasic  acid,  and  accordingly  combines  with  bases  to  form  neutral 
and  acid  salts.  Of  these,  the  neutral  salts  of  potassium  and 
lithium  are  the  most  soluble,  while  the  acid  salts,  and  notably  acid 
ammonium  urate,  are  quite  insoluble.  Its  compounds  with  the 
alkaline  earths  are  likewise  soluble  only  with  great  difficulty.  In 
the  urine  uric  acid  is  said  to  be  present  as  a  quadriurate,  viz.,  as 
a  hyperacid  compound,  in  which  one  molecule  of  sodium  (viz., 
potassium  or  ammonium)  is  in  combination  with  two  molecules  of 
uric  acid.  Its  solubility  in  the  urine  is  largely  dependent  upon  the 
amount  of  water,  the  reaction,  and  the  presence  of  mineral  salts  and 
possibly  of  pigments.  On  standing,  however,  in  the  absence  of 
micro-organisms,  the  quadriurate  is  decomposed,  with  the  liberation 
of  free  uric  acid  and  acid  biurates,  which  latter  are  then  again  trans- 
formed into  quadriurates  through  the  agency  of  the  diacid  phos- 
phates, and  through  a  repetition  of  this  process  all  the  uric  acid 
finally  separates  out,  as  is  shown  in  the  equations : 

(1)  HNa.C5H2N403.C5H4N403  +  H20  =  C5H4N403  +  HNa.C5H2N403 

Sodium  quadriurate.  Uric  acid.  Acid  sodium 

biurate. 

(2)  2HNa.C5H2N403  +  NaH2P04       =  HNa.C5H2N403.C5H4N403  +  Na2HP04 

Acid  biurate.  Quadriurate. 

In  the  urine  of  birds  and  reptiles  the  uric  acid  is  said  to  occur 
exclusively  in  the  form  of  quadriurates.  Neutral  urates  are  not 
found  in  the  urine.  Of  the  compounds  which  uric  acid  forms  with 
the  salts  of  the  heavy  metals,  the  silver  and  copper  salts  deserve 
especial  mention,  as  some  of  the  methods  which  are  employed  in  the 
quantitative  estimation  of  the  substance  are  dependent  upon  their 


THE   ORGANIC  CONSTITUENTS  OF  THE   URINE.  237 

formation.  The  salts  of  uric  acid  are  readily  decomposed  by  hydro- 
chloric acid,  and  on  standing  the  free  substance  crystallizes  out  from 
the  solution.  The  intimate  relation  which  exists  between  uric  acid 
and  the  xanthin  bases,  as  also  its  character  as  a  diureid,  has  already 
been  considered  (pages  SO  and  82). 

Tests  for  Uric  Acid. — Murexid  Test. — If  a  few  crystals  of  uric 
acid  are  evaporated  with  a  few  drops  of  concentrated  nitric  acid  on 
a  porcelain  plate,  a  yellow  or  brick-red  residue  remains.  On  cool- 
ing, a  drop  or  two  of  ammonia  are  added,  when  a  beautiful  purple- 
red  color  develops,  owing  to  the  formation  of  ammonium  purpurate 
(murexid).  If  now  an  excess  of  sodium  hydrate  solution  is  added, 
the  ammonium  salt  is  transformed  into  the  corresponding  sodium 
salt,  and  the  purple  red  passes  into  a  bluish  violet.  This  disappears 
on  heating  and  does  not  return  on  cooling  (compare  with  the  similar 
reaction  of  xanthin  and  guanin). 

Copper  Test. — A  few  crystals  of  uric  acid  are  dissolved  in 
sodium  hydrate  solution  and  treated  with  a  few  drops  of  Folding's 
solution.  On  heating,  white  urate  of  copper  separates  out.  If  more 
copper  solution  is  added,  a  partial  reduction  of  the  cupric  oxide 
occurs,  owing  to  the  formation  of  allantoin. 

DexxigEs'  Test. — If  uric  acid  is  transformed  into  alloxan  by 
means  of  nitric  acid,  and  the  excess  of  acid  is  carefully  evaporated, 
a  blue  color  results  on  treating  the  residue  with  a  few  drops  of 
concentrated  sulphuric  acid  and  commercial  benzol  containing 
thiophen. 

Schiff's  Test. — If  a  piece  of  filter-paper  is  moistened  with  a 
solution  of  nitrate  of  silver,  and  a  drop  of  a  solution  of  uric  acid  in 
sodium  carbonate  is  added,  a  brownish-black  color  develops,  owing 
to  reduction  of  the  oxide  of  silver.  In  the  presence  of  only  0.002 
milligramme  of  uric  acid  a  yellow  color  is  obtained. 

Isolation  of  Uric  Acid. —  (Jric  acid  is  most  conveniently  prepared 
from  the  excrements  of  snakes,  in  which,  as  has  been  stated,  it 
exists  in  the  form  of  the  quadri urate.  To  this  end,  the  material  is 
boiled  with  a  dilute  solution  of  sodium  hydrate  so  long  as  ammonia 
is  evolved,  when  carbon  dioxide  is  passed  through  the  solution  until 
the  alkaline  reaction  has  largely  disappeared.  The  acid  biurate  of 
sodium  which  separates  out  is  then  washed  with  eold  water  and 
dissolved  in  a  dilute  sodium  hydrate  solution.  On  adding  an 
-  of  concentrated  hydrochloric  acid  tin;  uric  acid  crystallizes 
out   on  standing. 

From  human  urine  the  substance  can  be  obtained  by  adding  con- 
centrated hydrochloric  acid  in  the  proportion  of  50  :  1000,  and 
keeping  the  mixture  at    a   low  temperature   for  about   forty-eight 

hours.      The    crystals    which     then    separate    out     are    treated     with 

water,  dissolved  in  dilute -odium  hydrate  solution,  decolorized  with 
animal  charcoal,  and  reprecipitated  with  hydrochloric  acid.  This 
method  was  formerly  employed  lor  estimating  the  amount  of  uric 
acid  in  the  urine,  but  has  dow  been  abandoned,  as  it  doe-  not  furnish 


238  THE   URINE. 

reliable  results  and  in  its  place  the  method  of  Hopkins  or  of 
.Ludwig-Salkowski  is  now  almost  exclusively  used  (see  below). 

Quantitative  Estimation. — Hopkins'  Method. — This  method 
furnishes  results  which  are  as  accurate  as  those  obtained  with  the 
older  method  of  Ludwig-Salkowski,  and  has,  above  all,  the  advan- 
tage of  greater  simplicity.  It  is  based  upon  the  fact  that  uric  acid 
can  be  completely  precipitated  from  the  urine  by  the  addition  of  cer- 
tain ammonium  salts.  Insoluble  acid  ammonium  urate  thus  results, 
which  is  transformed  into  the  free  acid,  and  this  estimated  either 
gravimetrically  or  by  titration  with  a  solution  of  potassium  per- 
manganate of  known  strength.  According  to  Folin's  most  recent 
modification  of  the  original  method,  we  may  proceed  as  follows  : 

Folin's  Method. — To  precipitate  the  uric  acid,  and  also  to  re- 
move the  small  amount  of  mucoid  substance  which  is  found  in 
every  urine,  the  following  reagent  is  employed  :  500  grammes  of 
ammonium  sulphate,  5  grammes  of  uranium  acetate,  and  60  c.c.  of  a 
10  per  cent,  solution  of  acetic  acid  are  dissolved  in  650  c.c.  of  water. 
The  resulting  solution  measures  about  1000  c.c.  Seventy-five  c.c.  of 
the  reagent  are  added  to  300  c.c.  of  urine  in  a  flask  holding  500  c.c. 
After  standing  for  five  minutes  the  mixture  is  filtered  through  two 
folded  filters,  and  thus  freed  from  the  mucoid  body,  which  is  carried 
■down  with  the  uranium  phosphate  in  acid  solution.  The  filtrate  is 
divided  into  two  portions  of  125  c.c.  each,  which  are  placed  in 
beakers  and  treated  with  5  c.c.  of  concentrated  ammonia.  After 
stirring  a  little  the  solutions  are  set  aside  until  the  next  day.  The 
supernatant  fluid  is  then  carefully  poured  off  through  a  filter 
(Schleicher  and  Schiill  No.  597) ;  the  precipitated  ammonium 
urate  is  collected  with  the  aid  of  a  small  amount  of  a  10  per 
cent,  solution  of  ammonium  sulphate  and  washed  with  the  same 
reagent.  Traces  of  chlorides  do  not  interfere  with  the  subsequent 
titration,  and  the  process  of  filtration  and  washing  can  be  completed 
in  from  twenty  to  thirty  minutes.  The  ammonium  urate  is  then 
washed  into  a  beaker,  after  opening  the  filter,  using  about  100  c.c. 
of  water.  Fifteen  c.c.  of  concentrated  sulphuric  acid  are  then  added 
and  the  solution  is  titrated  at  once  with  a  one-twentieth  normal  solu- 
tion of  potassium  permanganate.  Toward  the  end  of  the  titration 
Folin  suggests  to  add  the  permanganate  in  portions  of  two  drops  at 
a  time,  until  the  first  trace  of  rose  is  apparent  throughout  the  entire 
fluid.  Each  cubic  centimeter  of  the  reagent  corresponds  to  0.00375 
gramme  of  uric  acid.  A  final  correction  of  0.003  gramme  for  every 
100  c.c.  of  urine  employed  is  necessary,  owing  to  the  slight  degree 
to  which  ammonium  urate  is  soluble. 

Ludwig-Salkowski  Method. — This  is  based  upon  the  forma- 
tion of  insoluble  magnesium  silver  urate  when  urine  is  treated  with 
ammoniacal  magnesia  mixture  and  subsequently  with  an  ammoniacal 
solution  of  silver  nitrate,  while  the  chlorides  remain  in  solution. 
The  double  salt  is  then  decomposed  and  the  uric  acid  obtained  as 
such,  or  the  amount  of  silver  is  ascertained  by  titration  and  the  cor- 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  239 

responding  amount  of  uric  acid  calculated.  The  gravimetric  method 
is  probably  the  most  accurate.  The  titration  method  presupposes 
that  the  composition  of  magnesium-silver  urate  is  constant,  viz.,  that 
it  contains  one  molecule  of  uric  acid  for  every  atom  of  silver;  but 
this  has  not  been  definitely  established. 

For  clinical  purposes,  however,  the  titration  method  may  be  em- 
ployed, as  the  error  which  is  thus  involved  is  probably  only  slight. 

Method. — Two  hundred  and  fifty  c.c.  of  urine,  which  should 
present  a  specific  gravity  approximating  1.020,  arc  treated  with  50 
c.c.  of  ammoniacal  magnesia  mixture.  (This  is  prepared  by  dis- 
solving 100  grammes  of  magnesium  chloride  in  water,  adding  a  cold 
saturated  solution  of  ammonium  chloride  in  excess,  and  enough 
strong  ammonia  to  impart  a  decided  odor.  If  the  mixture  is  not 
olear,  more  of  the  ammonium  chloride  solution  is  added,  and  it  is 
finally  diluted  with  water  to  the  1000  c.c.  mark.)  After  filtering 
off  the  phosphates,  which  are  precipitated  by  the  magnesium 
mixture,  250  c.c.  of  the  filtrate,  corresponding  to  200  c.c.  of  urine, 
are  treated  with  20  c.c.  of  an  ammoniacal  3  per  cent,  silver  nitrate 
solution.  In  this  manner  the  uric  acid,  as  also  the  xanthin  bases, 
are  precipitated  as  silver  salts,  and  are  allowed  to  settle.  A  few 
cubic  centimeters  of  the  clear  supernatant  fluid  are  then  examined 
to  ascertain  whether  a  sufficient  quantity  of  the  silver  solution  has 
been  added.  To  this  end,  it  is  only  necessary  to  acidify  with  con- 
centrated nitric  acid,  when  a  precipitate  of  silver  chloride  should 
occur.  If  this  does  not  happen,  the  specimen  is  again  rendered 
alkaline  with  ammonia,  poured  back,  and  the  entire  volume  treated 
with  a  little  more  of  the  silver  solution. 

The  gelatinous  precipitate  is  filtered  off  with  the  aid  of  a 
suction-pump,  washed  free  from  chlorides  and  silver  with  weak 
ammonia-water,  and  transferred  to  a  beaker.  Twenty  c.c.  of  a 
solution  of  sodium  sulphide,  diluted  with  an  equal  volume  of  water, 
arc  brought  to  the  boiling-point,  and  then  immediately  added. 
(The  sulphide  solution  is  prepared  by  dissolving  10  grammes  of 
chemically  pure  sodium  hydrate  in  1000  c.c  of  distilled  water;  one- 
half  of  this  volume  is  saturated  with  hydrogen  sulphide  and  mixed 
with  thi-  other  half.)  A  little  more  boiling  water  is  added  to  the 
mixture,  which  is  kept  on  the  water-bath  for  a  while.  As  soon 
as  the  supernatant  fluid  is  perfectly  colorless  the  solution  is  allowed 
to  c»ol.  The  sulphide  of  <ilver  is  then  filtered  off  and  washed  with 
hot  water.  Filtrate  and  washings  are  then  acidified  with  hydro- 
chloric acid   and   evaporated  to  about   15  C.C. 

After  adding  :i  little  more  hydrochloric  acid  the  solution  is 
allowed  to  stand  for  twenty-four  hours,  when  the  uric  acid  is 
filtered    off,    washed    with    water,    then    with    alcohol,    and    dried    at 

115°  C.  For  every  10  c.c.  of  the  mother-liquor  and  water  used  in 

the  final  washing,  0.00048   gramme  i-  finally  added  to  the  result, 

to  allow  for  the  trifling  amount  of  uric  acid  which  remains  in 
solution. 


240  THE  URINE. 

Instead  of  decomposing  the  silver  compounds  as  described  with 
sodium  sulphide,  we  may  proceed  as  follows  :  the  precipitate  is 
suspended  in  about  300  c.c.  of  acidulated  water  and  subjected  to  a 
current  of  hydrogen  sulphide  until  the  decomposition  is  complete ; 
on  subsequent  boiling  all  the  uric  acid  passes  into  solution,  and  can 
be  separated  from  the  precipitate  of  silver  sulphide  by  filtration. 
Filtrate  and  washings  are  then  further  treated  as  described. 

Albumin  and  sugar,  if  present,  must  in  either  case  be  removed. 

The    Xanthin   Bases. 

The  xanthin  bases  which  have  been  found  in  the  urine  of  man 
are  xanthin,  hypoxanthin,  guanin,  carnin,  paraxanthin,  heteroxan- 
thin,  episarcin,  and  under  certain  pathological  conditions  adenin. 
Their  amount,  however,  is  always  small,  and  normally  constitutes 
about  10  per  cent,  of  the  quantity  of  uric  acid,  viz.,  from  0.02  to 
0.06  gramme.  Of  this  amount,  from  0.02  to  0.03  gramme  is  repre- 
sented by  xanthin.  Hypoxanthin  and  guanin  probably  stand  next 
in  order,  while  paraxanthin  and  heteroxanthin  are  found  only  in 
traces.  From  10,000  liters  of  urine  Kriiger  and  Salomon  thus 
obtained  only  7.5  grammes  of  the  latter. 

Origin. — It  has  been  shown  that  the  xanthin  bases  are  derived 
from  the  nuclear  nucleins,  and  are  probably  formed  in  all  the  tissues 
of  the  body.  There  is  reason  to  suppose,  moreover,  that  a  certain 
fraction  is  referable  to  ingested  nucleins.  Under  normal  conditions 
the  greater  portion  of  the  xanthin  bases  is  then,  no  doubt,  oxidized 
to  uric  acid,  but  a  variable  fraction  escapes  as  such.  To  a  certain 
extent  the  oxidation  to  uric  acid  occurs  in  the  liver,  but,  as  I  have 
shown,  this  takes  place  also  in  other  organs  of  the  body,  as  both 
xanthin  bases  and  uric  acid  are  obtained  in  transfusion  experiments. 
At  the  same  time  it  was  noted  that  the  relative  amount  of  the  two 
was  largely  influenced  by  the  degree  of  oxygenation  of  the  blood,  so 
that  xanthin  bases  only  were  obtained  if  venous  blood  was  used, 
Avhile  both  were  found  when  arterial  blood  was  employed.  We 
can  thus  understand  that,  as  a  general  rule,  at  least  a  certain  rela- 
tion exists  in  the  elimination  of  uric  acid  and  the  xanthin  bases. 
A  diminished  elimination  of  the  latter  is  thus  quite  frequently 
associated  with  a  corresponding  increase  of  the  former,  or  vice  versa, 
and  both  may,  of  course,  be  increased  or  diminished  together.  The 
most  notable  increase  in  their  elimination  is  observed  in  leuksemia, 
and  here  adenin  also  appears  in  the  urine. 

Theobromin  (dimethyl-xanthin)  and  caifein  (trimethyl-xanthin) 
are  partly  eliminated  in  the  urine  as  such,  and  partly  appear  as  a 
methyl-xanthin  which  is  apparently  identical  with  heteroxanthin. 

Xanthin  has  once  been  found  in  crystalline  form  in  a  urinary 
sediment,  and  has  in  several  instances  been  encountered  in  vesical 
calculi. 

As  the  isolation  of  the  individual  substances  from  the  urine  in 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  241 

amounts  sufficient  for  purposes  of  study  is  a  very  complicated  process, 
and  requires  facilities  which  are  not  generally  found  in  university 
laboratories,  it  will  suffice  at  this  place  to  describe  a  method  by 
which  they  can  be  collectively  estimated.  For  a  consideration  of 
the  chemical  properties  of  the  more  important  members  of  the  group 
and  their  isolation  from  other  sources,  as  also  of  their  relation  to 
uric  acid  and  the  nucleinic  bases,  the  reader  is  referred  to  other 
sections. 

Quantitative  Estimation. — The  xanthin  bases  are  best  isolated 
from  the  urine  according  to  Salkowski's  method,  which  is  based 
upon  their  precipitation  as  silver  compounds,  together  with  uric  acid, 
the  separation  of  the  latter,  and  the  determination  of  the  amount  of 
silver  in  combination  with  the  bases.  To  this  end,  600  c.c.  of 
urine  are  first  treated,  as  described  in  the  quantitative  estimation 
of  uric  acid,  according  to  Ludwig-Salkowski.  The  final  filtrate 
after  removal  of  the  uric  acid,  together  with  the  washings,  is  then 
treated  with  ammoniacal  silver  solution  and  the  xanthin  bases  thus 
reprecipitated.  The  precipitate  is  collected  on  a  small  filter,  washed 
with  water,  dried,  and  incinerated.  The  ash  is  dissolved  in  dilute 
nitric  acid,  and  the  silver  estimated  by  titrating  with  a  solution  of 
potassium  sulphocyanide  of  known  strength,  using  ammonio-ferric 
alum  as  an  indicator.  As  it  has  been  ascertained  that  in  an  equal 
mixture  of  the  silver  compounds  of  xanthin,  hypoxanthin,  guanin, 
etc.,  one  atom  of  silver  represents  0.277  gramme  of  nitrogen  or 
0.7381  gramme  of  the  bases,  1  c.c.  of  the  sulphocyanide  solution, 
that  is  commonly  used  in  the  estimation  of  the  chlorides  of  the 
urine  (page  219),  will  correspond  to  0.002  gramme  of  nitrogen  or 
0.00542  gramme  of  the  bases. 

The  method  of  Kriiger  and  Wulff,  which  was  greatly  in  vogue  a 
few  years  ago,  has  been  abandoned,  as  the  values  thus  obtained  were 
too  high.  According  to  this  method,  the  alloxuric  bodies,  viz.,  uric 
acid  and  xanthin  bases,  were  first  estimated  by  precipitating  with 
copper  sulphate  and  sodium  bisulphite  and  determining  the  amount 
of  nitrogen  in  the  precipitate.  In  a  second  portion  of  the  urine  the 
uric  acid  was  then  estimated  and  the  corresponding  amount  of 
nitrogen  deducted  from  the  first  result.  The  difference  was  referred 
to  the  xanthin  bases. 

Oxalic  Acid  and  Oxaluric  Acid. 

Of  the  origin  of  oxalic  acid  and  oxaluric  acid,  both  of  which  may 
oe  regarded  as  normal  constituents  of  the  urine,  but  little  is  known. 
The  former  i-  supposedly  presenl  ;is  a  calcium  suit,  which  is  held 
in  solution  owing  to  the  presence  of  diacid  sodium  phosphate,  but 
readily  separates  out  on  standing  and  is  then  frequently  encountered 
in  urinary  sediments.  Here  ii  generally  occurs  in  the  very  charac- 
teristic envelope  or  dumb-bell  firms,  and  can  be  readily  distin- 
guished  from  other  constituents   by   its  insolubility  in  acetic  acid, 

16 


242  THE   URINE. 

and  its  solubility  in  hydrochloric  acid.  Its  amount  normally  varies 
between  0.02  and  0.05  gramme.  Oxaluric  acid,  on  the  other  hand, 
exists  in  the  urine  as  an  ammonium  salt  and  is  not  found  in  sedi- 
ments.    Its  amount  is  even  smaller  than  that  of  oxalic  acid. 

As  many  articles  of  food,  such  as  asparagus,  spinach,  grapes, 
apples,  etc.,  contain  oxalic  acid  in  not  inconsiderable  amounts,  it  is 
supposed  that  a  certain  fraction  of  the  oxalic  acid  of  the  urine  is 
referable  to  this  source.  We  find,  as  a  matter  of  fact,  that  in  the 
asparagus  season  larger  amounts  are  eliminated  than  at  any  other 
time  of  the  year.  But  it  has  also  been  noted  that  oxalic  acid  does 
not  disappear  from  the  urine  when  the  diet  consists  exclusively  of 
albumins  and  fats,  and  that  during  starvation  also  oxalic  acid  can 
still  be  found.  We  are  consequently  forced  to  the  conclusion  that 
a  certain  amount  of  the  substance  must  originate  in  the  tissues  of 
the  body,  and  there  is  a  growing  belief  that  the  albumins  are  here 
its  ultimate  source.  We  know  indeed  that  oxaluric  acid  is  closely 
related  to  uric  acid,  and  it  in  turn  can  be  decomposed  into  urea  and 
oxalic  acid,  as  is  shown  by  the  equations : 


(1)  C5H4N403  +  O  +  H20 
Uric  acid. 


CO  —  NH 


(2)  CO  >CO+0 

co  —  nh/ 

Alloxan. 

CO  —  NH  CO  —  NH^ 

(3)  |  >CO  +  H20  =   |  >CO 
CO  — NHX                                 COOH-NH/ 

Parabanic  acid.  Oxaluric  acid. 

CO  -  NH^  CO  —  OH  7NH2 

(4)  I  ^CO  +  H20=   I  +CO< 
COOH-NH/                      CO -oh  \nh2 

Oxaluric  acid.  Oxalic  acid.  Urea. 

As  oxalic  acid  on  further  oxidation  is  decomposed  into  water  and 
carbon  dioxide,  it  would  thus  appear  that  both  oxaluric  acid  and 
oxalic  acid  may  be  regarded  as  complete  oxidation-products  of  uric 
acid.  We  find,  as  a  matter  of  fact,  that  oxalic  acid  is  increased  in 
various  diseases  in  which  the  oxidation-processes  are  manifestly 
impaired,  such  as  diabetes  mellitus,  various  diseases  of  the  circulatory 
apparatus  when  associated  with  deficient  oxygenation  of  the  blood, 
in  obesity,  etc.  I  have  frequently  observed,  moreover,  that  an 
increased  elimination  of  oxalic  acid  is  associated  with  an  increased 
excretion  of  uric  acid  in  young,  more  or  less  ansemic  individuals  of 
a  neurotic  type.  Whether  or  not  oxalic  acid  may  further  be  derived 
from  carbohydrates  is  as  yet  unknown,  but  is  rather  improbable. 


CO- 

1 

■NHv 

1 
CO 

1 

\C0  +  CO(NH2). 

co  —  nh/ 

Alloxan. 

CO  —  NH, 

|                 >CO  +  C02 
CO  —  NH^ 

Parabanic  acid. 

THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  243 

Aside  from  its  occurrence  in  solution  and  in  urinary  sediments,  oxalic 
acid  is  also  not  infrequently  found  to  constitute  the  greater  portion 
of  renal  and  vesical  calculi. 

Quantitative  Estimation  of  Oxalic  Acid. — Dunlop's  Method 
(modified  by  Baldwin). — Five  hundred  cc.  of  a  well-mixed  specimen 
of  the  twenty-four  hours'  urine  are  treated  with  150  cc.  of  over  90 
per  cent,  alcohol,  to  precipitate  the  oxalate  of  calcium.  After  forty- 
eight  hours  the  crystals  are  collected  on  a  filter,  thoroughly  washed 
with  hot  and  cold  water  and  with  dilute  acetic  acid  (1  per  cent.).  The 
filter  is  placed  in  a  small  beaker  and  soaked  in  a  small  amount  of 
dilute  hydrochloric  acid.  It  is  then  washed  with  hot  water  until 
there  is  no  further  acid  reaction.  The  washings  are  filtered  and 
evaporated  to  about  20  cc.  A  very  little  calcium  chloride  solution 
is  added  to  insure  an  excess  of  calcium.  The  hydrochloric  acid  is 
neutralized  with  ammonia  and  the  solution  then  rendered  slightly 
acid  with  acetic  acid.  Strong  alcohol  is  now  added  to  the  amount 
of  50  per  cent,  of  the  volume  of  the  fluid  and  the  solution  set  aside 
for  forty-eight  hours.  The  sediment  is  collected  on  a  filter  that  is 
free  from  ash,  and  washed  with  cold  water  and  dilute  acetic  acid  until 
free  from  chlorides.  (Hot  water  should  here  be  avoided,  as  it  carries 
the  finely  divided  precipitate  through  the  pores  of  the  filter.)  The 
filter  is  incinerated  over  a  Bunsen  burner,  and  afterward  heated  in 
the  blowpipe-flame.  The  residue  is  allowed  to  cool  over  sulphuric 
acid  and  weighed.  The  ash  is  calcium  oxide,  each  gramme  of  which 
corresponds  to  1.6  grammes  of  oxalic  acid. 

The  urine  in  every  case  should  be  thymolized  as  soon  as  possible, 
to  prevent  fermentation  and  the  precipitation  of  phosphates.  If  the 
specimen  is  alkaline,  it  is  rendered  slightly  acid  with  acetic  acid. 

The  method  is  applicable  in  the  case  of  human  urine,  but  in 
that  of  dog-  with  a  high  specific  gravity  it  is  very  difficult  to  remove 
the  phosphate-.  In  such  an  event  Salkowski's  method  is  best 
employed. 

OALKOWSKl's  METHOD. — If  the  urine  is  concentrated  (sp.  gr. 
1.040-1.050),  it  is  treated  with  20  cc  of  hydrochloric  acid  (sp.  gr. 
1.12)  fin'  200-250  cc,  and  extracted  in  a  separating  funnel  three 
times  with  alcoholic  ether  (5-10  per  cent,  alcohol).  Tlje  ethereal 
extract  is  filtered  through  a  dry  filter,  the  ether  is  distilled  off,  the 
remaining  fluid  evaporated  to  20  cc,  and  filtered  on  cooling.  The 
filtrate  i-  rendered  alkaline  with  ammonia,  and  is  then  treated  with 
1-2  e.e.  of  a  10  per  cent,  solution  of  calcium  chloride  and  acetic 
acid.     The  process  is  continued  as  described. 

With  human  urine  Larger  quantities,  such  as  500  e.e.,  are  em- 
ployed, which  an;  first  concent  ruled  to  about  one-third  of  their 
original   volume. 

Allantoin. 

Allantoic    i-    a    normal    constituent    of  the    urine   of  man,  as  also 
of    various    animals,    but    i-    usually    present    only    in  traces   in    the 


244       ■  THE  URINE. 

adult,  while  during  the  first  weeks  of  life  it  is  more  abundant. 
Larger  amounts  also  occur  during  pregnancy.  Aside  from  the 
urine,  it  is  found  in  the  amniotic  fluid  and  in  the  allantoic  fluid  of 
cows.  Like  oxaluric  acid,  it  is  an  oxidation-product  of  uric  acid, 
and  Salkowski  demonstrated  that  an  increased  elimination  occurred 
in  dogs  when  uric  acid  was  ingested.  An  artificial  increase  is 
similarly  observed  in  poisoning  with  hydrazin,  and  we  can  readily 
understand  that  in  consequence  of  the  degenerative  changes  which 
are  thus  produced  in  the  liver  the  oxidation-processes  are  accordingly 
diminished,  and  allantoin  results. 

The  formation  of  allantoin  from  uric  acid  may  be  represented  by 
the  equation : 

.NH.CH.NH.CO.NH2 
C5H4NA  +  H20  +  O  =  CO''  +  C02 

xNH.CO 
Uric  acid.  Allantoin. 

It  is  a  glyoxyl-diureid,  and  may  be  produced  artificially  by 
heating  glyoxylic  acid  and  urea  together  at  a  temperature  of  100°  C. 
The  reaction  then  takes  place  according  to  the  equation  : 

/NH2         COH  ,NH2  /NH.CH.NH.CO.NH2 

CO<  +     |  +    CO<         '    =    CO(  +    2H20 

\NH2         COOH  XNH2  xNH.CO 

Urea.        Glyoxylic  Urea.  Allantoin. 

acid. 

The  substance  crystallizes  in  colorless  rhombic  prisms,  which  are 
frequently  grouped  in  the  form  of  stars  and  rosettes.  It  is  soluble 
with  some  difficulty  in  cold  water,  more  readily  in  hot  water  and 
hot  alcohol,  while  in  cold  alcohol  and  ether  it  is  insoluble.  On  pro- 
longed boiling,  it  reduces  Fehling's  solution,  and  it  is  owing  to  the 
formation  of  this  substance,  as  already  indicated,  that  uric  acid  can 
reduce  cupric  oxide  when  boiled  in  alkaline  solution  (see  page  237). 
With  silver  nitrate  allantoin  forms  a  crystalline  precipitate  of  allan- 
toin silver,  if  ammonia  is  cautiously  added  to  the  solution,  but  it  is 
soluble  in  an  excess  of  the  alkalies. 

Isolation. — Allantoin  is  most  conveniently  obtained  from  the 
urine  of  calves.  To  this  end,  the  urine  is  evaporated  on  a  water- 
bath  to  a  thick  syrup  and  allowed  to  stand  for  several  days  in  the 
cold.  The  crystalline  constituents  which  have  formed  are  then 
separated  mechanically  from  the  mother-liquor  and  the  gelatinous 
material  which  is  present,  and  dissolved  in  a  small  amount  of  hot 
water.  The  solution  is  decolorized  with  animal  charcoal,  filtered 
while  hot,  and  the  filtrate  acidified  slightly  with  hydrochloric  acid. 
On  standing,  the  allantoin  separates  out. 

Kreatinin. 

The  kreatinin  which  is  found  in  the  urine  of  man  and  all 
mammals  is  in  all  probability  derived  from  the  kreatin  of  the 
muscle-tissue,  and  is  thus  in  part  referable  to  the  ingested  animal 


THE  ORGANIC  CONSTITUENTS  OF  THE   URINE.  245 

food,  and  in  part  to  the  wear  and  tear  which  constantly  goes  on  in 
the  muscular  structures  of  the  body.  The  latter  source,  as  com- 
pared with  the  former,  is  normally,  however,  of  secondary  impor- 
tance, as  the  greater  amount  of  kreatin  which  originates  in  the 
muscles  is,  in  health  at  least,  transformed  into  urea.  We  accord- 
ingly find  that  the  elimination  of  kreatin  is  much  reduced  when  the 
animal  is  placed  on  a  diet  of  milk  exclusively,  while  it  is  increased 
if  a  liberal  amount  of  meat  is  ingested,  or  kreatin  is  administered 
as  such.  On  the  other  hand,  we  observe  that  muscular  exercise  in 
itself  does  not  call  forth  an  increased  excretion,  although  this  has 
recently  been  denied.  Under  pathological  conditions,  however,  in 
which  an  increased  destruction  of  the  albuminous  constituents  of  the 
body  occurs,  abnormally  large  quantities  may  be  found  in  the  urine, 
although  the  patient  receives  food  which  is  free  from  kreatin.  In 
such  cases  we  may  assume  that  the  muscles  have  lost  to  a  greater  or 
less  degree  the  power  of  transforming  kreatin  into  urea.  Kreatin 
itself  has  thus  far  not  been  found  in  perfectly  fresh  urine,  but  is 
formed  from  the  kreatinin  on  the  occurrence  of  bacterial  decomposi- 
tion. The  transformation  of  kreatin  into  its  anhydride  supposedly 
occurs  in  the  kidneys,  and  we  accordingly  find  that  in  extensive 
disease  of  these  organs  the  elimination  of  kreatinin  is  diminished. 

Of  late,  it  has  been  claimed  that  the  kreatinin  which  is  found  in 
muscle-tissue  is  not  identical  with  the  urinary  kreatinin,  and  it  has 
even  been  suggested  that  the  source  of  the  latter  may  have  to  be 
sought  in  other  organs  of  the  body,  and  notably  in  the  thyroid 
gland,  in  which  kreatinin  has  been  found  in  considerable  amount. 
It  is,  of  course,  possible  that  the  substance  may  be  formed  in  other 
organs  as  well,  but  the  evidence  is  conclusive  that  the  urinary  form 
is  largely  derived  from  muscle-tissue.  But  even  supposing  that 
future  researches  should  show  that  the  two  bodies  are  isomeric,  but 
not  identical,  we  could  even  then  imagine  that  both  originate  from 
the  same  substance. 

The  amount  of  kreatinin  which  is  normally  found  in  the  urine  of 
man  is  approximately  1  gramme,  but  varies,  of  course,  with  the 
character  of  the  food. 

Properties. — Kreatinin  crystallizes  in  colorless,  highly  refractive 
monoclinic  prisms,  and  is  quite  soluble  in  hot  and  cold  water,  less 
readily  so  in  alcohol,  and  is  insoluble  in  ether.  In  aqueous  solution 
it  i-  gradually  transformed  into  kreatin.  The  same  transformation 
results  more  rapidly  when  the  substance  i<  heated  in  alkaline  solu- 
tion. It  combines  with  acids  and  various  salts  to  form  crystalline 
compounds,  some  of  which  are  characteristic.  This  is  true  especially 
of  the  chlorozincatt — (C^HyNjOg^.Zni  !12 — which  results  when  a  con- 
centrated alcoholic  solution  of  kreatinin  is  treated  with  a  solution 
of  zinc  chloride,  which  should  be  as  little  acid  as  possible.  The 
crystalline  form  of  this  compound  depends  very  much  upon  its 
purity.      As   firsi    obtained    from   the   urine,  it  occurs  in  the  form  of 

varicose  conglomerations,  which  usually  adhere  firmly  to  the  walls 


246  THE   URINE. 

of  the  vessel.  In  pure  form  it  crystallizes  in  fine  needles,  which 
are  commonly  grouped  in  sheaves  and  stars.  The  salt  is  almost 
insoluble  in  alcohol,  and  nearly  so  in  water,  while  free  mineral 
acids  dissolve  it. 

Kreatinin  is  further  precipitated  by  mercuric  nitrate,  notably  in 
the  presence  of  sodium  carbonate,  by  silver  nitrate,  platinum 
chloride,  stannous  chloride,  mercuric  chloride,  picric  acid,  phospho- 
tungstic  acid,  etc.  With  all  these  substances  it  forms  well-charac- 
terized crystalline  salts.  In  alkaline  solution  it  reduces  cupric 
hydroxide,  and  it  is  for  this  reason  that  urines  which  contain  much 
kreatinin  but  no  sugar  give  a  positive  reaction  with  Fehling's 
solution.  The  separation  of  the  cupric  oxide,  however,  only  occurs 
on  prolonged  boiling.  An  alkaline  solution  of  bismuth,  on  the 
other  hand,  is  not  affected. 

Tests. — Weyl's  Test. — A  few  cubic  centimeters  of  urine  are 
treated  with  a  few  drops  of  a  freshly  prepared,  very  dilute  solution 
of  sodium  nitroprusside,  and  then  drop  by  drop  with  a  solution 
of  sodium  hydrate,  when  in  the  presence  of  kreatinin  a  ruby-red 
color  develops,  which  is  especially  pronounced  in  the  lower  portion 
of  the  tube.  After  a  few  minutes  the  color  disappears.  If  now 
acetic  acid  is  added  in  excess  and  the  mixture  heated,  it  assumes  a 
greenish  color,  then  turns  blue,  and  on  standing  deposits  a  sediment 
of  Prussian  blue.  Acetone  and  diacetic  acid  give  a  similar  reaction 
if  ammonia  is  added  instead  of  sodium  hydrate ;  but  with  kreatinin 
no  red  color  is  thus  obtained. 

Jaffe's  Test. — If  a  few  cubic  centimeters  of  urine  are  treated 
with  a  dilute  aqueous  solution  of  picric  acid,  the  corresponding 
compound  of  kreatinin  is  precipitated.  On  adding  a  few  drops  of 
a  dilute  solution  of  sodium  hydrate  a  red  color  develops,  which  per- 
sists for  several  hours,  and  is  changed  to  yellow  upon  the  addition 
of  an  acid.  With  glucose  a  red  color  also  is  obtained,  but  only  on 
heating,  while  the  reaction  in  the  case  of  kreatinin  takes  place  only 
at  ordinary  temperatures.     Acetone  gives  a  reddish-orange  color. 

Synthesis  of  Kreatinin. — Kreatinin  can  be  formed  synthetically 
from  methyl-glycocoll  and  cyanamide.  Kreatin  is  first  produced, 
and  then  yields  the  anhydride,  as  shown  in  the  equations  : 

NH 

(1)  N=C  —  NH2  +  XH(CH3)CH2.COOH  =  NH=C< 

X(CH3).CH2.COOH 

Cyanamide.  Methyl-glycocoll.  Kreatin. 

XNH2  yNH CO 

(2)  NH=C<  =  NH=C<  |       +  H20 

XN(CH3).CH2.COOH  XN(CH3).CH2 

Kreatin.  Kreatinin. 

Isolation  and  Quantitative  Estimation. — The  quantitative  estima- 
tion of  kreatinin  in  the  urine  is  based  upon  the  formation  of  the 
chlorozincate,  which  is  almost  insoluble  in  alcohol :  240  c.c.  of 
urine,  which  should  be  free   from  albumin  and  sugar,  are  rendered 


THE  AROMATIC  CONSTITUENTS  OE  THE   URINE.         247 

alkaline  with  milk  of  lime,  and  treated  with  a  solution  of  calcium 
chloride  so  long  as  a  precipitate  forms.  Water  is  then  added  to  the 
.')()<)  c.c.  mark.  After  standing  for  a  while  the  phosphates  are 
tillered  off.  The  precipitate  is  washed  with  a  little  water.  Filtrate 
and  washings  are  rendered  slightly  aeid  with  acetie  acid,  and  are 
then  evaporated  to  a  syrup.  This  is  stirred  while  still  warm  with 
about  25  to  30  C.C.  of  absolute  alcohol,  transferred  to  a  glass- 
stoppered  flask,  and  diluted  with  absolute  alcohol  to  100  c.c.  After 
twenty-four  hours  the  mixture  is  filtered.  The  filtrate  is  treated 
with  a  small  amount  of  sodium  acetate  in  solution,  and  is  con- 
centrated to  about  50  c.c.  To  this  is  added  0.5  c.c.  of  a  concentrated 
alcoholic  solution  of  zinc  chloride,  which  is  prepared  by  dissolving 
a  small  amount  of  the  salt  in  SO  per  cent,  alcohol,  and  diluting  with 
95  per  cent,  alcohol  to  a  specific  gravity  of  1.2.  The  mixture  is 
then  well  stirred  and  set  aside  in  a  cool  place  for  several  days. 
The  crystals  of  the  chlorozincate  of  kreatinin  are  now  collected 
on  a  previously  weighed  filter  and  washed  with  alcohol  until  free 
from  chlorides,  when  they  are  dried  at  100°  0.  and  weighed. 
The  corresponding  amount  of  kreatinin  is  ascertained  by  multiply- 
ing the  weight  by  0.6243.  The  material  which  is  thus  obtained 
is,  however,  always  impure,  owing  to  an  admixture  of  various  pig- 
ments and  traces  of  chlorides.  If  greater  accuracy  is  required,  it  is 
hence  necessary  to  determine  the  amount  of  zinc  in  the  crystals, 
which  may  be  done  as  follows:  the  material  is  covered  with  a 
little  nitric  acid  ;  the  solution  is  evaporated,  the  residue  incinerated, 
extracted  with  water,  ami  the  aqueous  solution  evaporated,  the 
residue  ignited,  and  finally  weighed.  The  zinc  is  thus  obtained  as 
oxide,  from  which  the  corresponding  amount  of  kreatinin  is  cal- 
culated by  multiplying  by  2.77DO. 

To  isolate  the  kreatinin  from  the  chlorozincate,  the  latter  is  dis- 
solved in  a  small  amount  of  hot  water  and  boiled  for  ten  minutes 
with  well-washed  plumbic  hydrate.  After  filtering  off  the  insoluble 
oxide  of  zinc  and  the  chloride  of  lead  the  filtrate  is  evaporated  to 
dryness  and  extracted  with  cold  absolute  alcohol.  This  takes  up 
the  kreatinin,  while  a  small  amount  of  kreatin  formed  during  the 
process  of  boiling  remains.  On  evaporation  the  kreatinin  is  obtained 
in  crystalline  form,  and  can  be  further  purified  by  recrystallization 
from  water. 

THE  AROMATIC  CONSTITUENTS  OF  THE  URINE. 

It  has   been    pointed  out    in    a    preceding  chapter   that   (luring  the 

process  of  intestinal  putrefaction  various  aromatic  bodies  are  formed 
from  the  albumins  of  the  food  or  their  products  of  digestion,  and 
are  then  absorbed  and  eliminated  in  the  urine,  either  as  such  or  in 
combination  with  sulphuric  acid,  glucuronic  acid,  or  glycocoll. 
Some  of  i Ik-,,  bodies,  Buch  :i^  indol  and  skatol,  may  be  regarded  as 
specific   product-  of   putrefaction  ;   while  others  or  closely   related 


248  THE   URINE. 

substances  occur  preformed  also  in  many  articles  of  food.  We  con- 
sequently recognize  two  sources  of  the  aromatic  bodies  which  are 
found  in  the  urine,  viz.,  the  aromatic  bodies  which  enter  into  the 
composition  of  our  diet  as  such,  and  those  which  result  from  the 
destruction  of  albumins  through  the  activity  of  micro-organisms. 
Under  certain  pathological  conditions,  further,  substances  of  this 
character  may  also  be  formed  in  the  body  proper,  owing  to  degenera- 
tive changes,  which  may  or  may  not  be  the  result  of  bacterial  action. 
Under  normal  conditions,  however,  this  source  scarcely  enters  into 
consideration. 

The  Conjugate  Sulphates. 

Paracresol,  phenol,  hydroquinon,  pyrocatechin,  indol,  and  skatol 
are  largely  eliminated  in  the  urine  in  combination  with  sulphuric 
acid  as  sodium  or  potassium  salts.  But  while  paracresol  and  its 
derivatives  combine  with  sulphuric  acid  directly,  indol  and  skatol 
are  previously  oxidized  to  indoxyl  and  skatoxyl,  as  has  been  shown. 
Conjointly  the  resulting  compounds  are  spoken  of  as  the  conjugate 
or  ethereal  sulphates  of  the  urine.  Their  daily  excretion  in  man 
corresponds  to  about  one-tenth  of  the  mineral  sulphates,  viz.,  from 
0.094  to  0.620  gramme,  under  normal  conditions.  Increased 
amounts  are  observed  when  from  whatever  cause  the  intestinal 
putrefaction  is  increased.  It  is  to  be  noted,  however,  that  the  ratio 
between  the  individual  substances  is  even  normally  not  constant, 
and  it  seems  that  the  relative  preponderance  of  the  one  over  the 
other  is  primarily  referable  to  the  extent  to  which  individual  groups 
of  micro-organisms  are  active.  In  some  instances  we  may  thus  find 
that  the  increase  in  the  conjugate  sulphates  is  referable  to  an  increased 
production  of  indol  and  .skatol,  while  in  others  phenols  are  largely 
formed. 

Aside  from  an  increase  in  the  degree  of  intestinal  putrefaction, 
larger  amounts  of  the  conjugate  sulphates  may  also  be  observed  if 
putrefactive  processes  are  taking  place  within  the  body  proper,  pro- 
viding that  active  resorption  occurs  from  the  diseased  area.  An 
increased  elimination  is  also  noted  when  any  of  the  aromatic  sub- 
stances mentioned  are  ingested  as  such  or  otherwise  introduced  into 
the  circulation  from  without.  A  notable  increase  is  thus  observed 
in  poisoning  with  carbolic  acid  or  its  congeners,  and  is  then,  of 
course,  principally  owing  to  an  increased  formation  of  phenol  sul- 
phates. The  ingestion  of  ortho-nitro-phenyl-propiolic  acid,  which 
is  reduced  to  indoxyl  within  the  body,  similarly  leads  to  an  increased 
elimination  of  indoxyl  sulphate. 

The  synthesis  of  the  various  conjugate  sulphates  is  probably 
effected  within  the  liver,  but  may  also  occur  in  other  organs  of  the 
body.     Their  quantitative  estimation  in  toto  has  been  described. 

The  Phenols. — Of  the  phenols  which  occur  in  the  urine,  para- 
cresol is  the  most  abundant ;  next  in  order  comes  phenol,  while 
pyrocatechin  and  hydroquinon  are  found  only  in  traces.     Besides 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         249 

paracresol,  the  normal  urine  of  man  is  said  also  to  contain  minute 
amounts  of  meta-  and  ortho-cresols.  The  total  elimination  of 
cresols  and  phenols,  however,  normally  corresponds  to  only  about 
0.03  gramme    in    the    twenty-four    hours. 

Urine  which  contain-  much  hydroquinon  or  pyrocatechin  gradually 
assumes  a  dark-brown  color  on  standing  if  the  reaction  is  alkaline, 
and  it  is  noted  that  this  change  in  color  begins  in  the  upper  layers 
and  gradually  extends  downward.  Ultimately  the  urine  becomes 
almost  black.  This  change  is  referable  to  oxidation  of  the  dioxy- 
benzols,  but  of  the  resulting  compounds  nothing  is  known.  Urines 
of  this  character  are  mostly  observed  in  poisoning  with  carbolic  acid, 
following  the  administration  of  benzol,  of  phenol  sulphates,  etc. 

To  demonstrate  the  presence  of  phenol  or  of  paracresol,  1000 
c.c.  of  urine  are  treated  with  70  c.c.  of  concentrated  hydrochloric 
acid,  and  distilled  until  about  one-fourth  of  the  total  amount 
has  passed  over.  The  conjugate  sulphates  are  thus  decomposed, 
and  phenol  and  cresol  are  found  in  the  distillate.  Their  presence 
can  here  be  demonstrated  by  testing  with  Millon's  reagent  or  by 
adding  bromine-water,  when  tribromophenol  crystallizes  out  on 
standing.  If  much  phenol  is  present,  it  may  further  be  possible 
to  obtain  a  positive  reaction  with  ferric  chloride  solution  if  this  is 
added  drop  by  drop  in  very  dilute  solution.  Hydroquinon  and 
pyrocatechin  remain  in  the  acid  solution.  To  demonstrate  their  pres- 
ence, the  solution  is  evaporated  to  about  100  c.c,  and  on  cooling  is 
extracted  with  an  equal  volume  of  ether.  Hydroquinon  and  pyro- 
catechin together  with  the  aromatic  oxy-acids  are  thus  removed. 
On  adding  a  dilute  solution  of  sodium  carbonate  to  the  ethereal  solu- 
tion the  aromatic  oxy-acids  are  transformed  into  the  corresponding 
sodium  salts.  The  ethereal  extract,  which  now  contains  only  the 
dioxy-benzols,  i-  evaporated  to  dryness  ;  the  residue  is  dissolved  in 
a  little  water  and  examined  as  follows:  one  portion  is  treated  drop 
by  drop  with  a  dilute  solution  of  ferric  chloride,  when  in  the  pres- 
ence of  pyrocatechin  a  green  color  develops,  which  turns  to  violet 
upon  the  addition  of  a  small  amount  of  tartaric  acid  and  ammonia. 
Tin-  remainder  of  the  solution  is  precipitated  with  lead  acetate  and 
filtered.  The  filtrate  contains  the  hydroquinon,  while  the  pyro- 
catechin is  in  the  precipitate.  The  hydroquinon  can  then  lie  isolated 
by  acidifying  and  extracting  with  ether,  when  the  substance  crys- 
tallizes out  on  evaporation.  It  is  dissolved  in  a  little  water,  and 
treated  drop  by  drop  with  the  dilute  iron  solution.  Quinon, 
C  I  !/>..  results,  and  may  be  recognized  by  it-  penetrating  odor. 

Quantitative  Estimation. — METHOD  of  KOSSLEB  and  PENNY, 
modified  by  Neuberg. — This  method  is  based  upon  the  precipi- 
tation of  phenol  and  paracresol,  by  mean-  of  iodine,  a-  tri-iodo- 
phenol.  prom  the  amount  of  iodine  which  is  thus  used  the  corre- 
sponding amount  of  monoxy-benzols  can  be  calculated,  and  is 
expressed  either  in  terms  of  phenol  or  cresol,  as  the  method  does 
not  indicate  the  separate  amount  of  the  individual   bodies  that  are 


250  THE    URINE. 

present.     The  reaction  which  takes  place  may  be  represented  by 
the  equation  : 

C6H5.OH  +  61  =  C6H2.I3OH  +  3HI. 

Five  hundred  c.c.  of  urine  are  rendered  feebly  alkaline  an.d 
evaporated  to  about  100  c.c.  Any  acetone  which  may  have  been 
present  is  thus  removed.  The  residual  fluid  is  acidified  with  sul- 
phuric acid,  so  as  to  contain  5  per  cent,  of  the  original  volume,  and 
is  then  repeatedly  distilled.  The  individual  portions  thus  obtained 
are  shaken  with  calcium  carbonate  until  the  acid  reaction  has  dis- 
appeared, so  as  to  remove  any  nitrous  or  formic  acid  that  may  be 
present.  The  fluid  is  now  again  distilled,  and  the  distillate  treated 
with  a  solution  of  1  gramme  of  caustic  soda  and  6  grammes  of  lead 
acetate  in  substance.  The  mixture  is  kept  on  a  boiling  water-bath 
for  about  fifteen  minutes.  A  portion  of  the  lead  oxide  is  thus  dis- 
solved by  the  phenol  to  form  basic  phenolates,  while  any  aldehydes 
or  ketones  that  may  have  been  formed  from  the  small  amount  of 
carbohydrates  that  are  present  in  every  urine  escape.  To  remove 
these  entirely,  the  mixture  is  heated  over  a  free  flame  connected  with 
a  condenser  until  a  few  cubic  centimeters  of  the  distillate  no  longer 
reduce  an  alkaline  solution  of  silver  nitrate.  After  five  minutes 
this  point  is  usually  reached.  The  fluid  is  then  acidified  with  sul- 
phuric acid  as  before,  and  is  distilled,  water  being  added  from  time 
to  time.  The  distillate  is  placed  in  a  glass-stoppered  bottle,  treated 
with  a  deci normal  solution  of  sodium  hydrate  until  the  reaction  is 
markedly  alkaline,  and  immersed  in  hot  water.  To  the  hot  fluid  a 
decinormal  solution  of  iodine  is  added  in  an  amount  which  should 
exceed  that  of  the  alkali  solution  by  15  to  25  c.c.  The  bottle  is 
now  closed  at  once,  shaken,  and  set  aside  until  cool.  The  solution 
is  then  acidified  with  dilute  sulphuric  acid,  and  the  excess  of  iodine, 
which  was  not  used  in  the  formation  of  tri-iodophenols,  retitrated  with 
a  decinormal  solution  of  sodium  thiosulphate.  One  c.c.  of  the  iodine 
solution  represents  1.567  milligramme  of  phenol,  or  1.8018  milli- 
gramme of  cresol.  As  the  latter  predominates  in  the  urine,  it  is  best 
to  express  the  results  in  terms  of  cresol.  Thus  modified,  the  method 
is  also  applicable  in  the  presence  of  sugar.  The  older  gravimetric 
method,  by  which  the  phenols  were  isolated  as  bromine  substitution- 
products,  has  now  been  largely  abandoned,  as  it  has  been  shown 
that  the  resulting  precipitate  is  not  of  constant  composition,  and 
contains  variable  amounts  of  dibromocresol,  besides  tribromophenol 
and  bromo-tribromophenol. 

Indoxyl  Sulphate. — The  incloxyl  sulphate  which  occurs  in  the 
urine  in  combination  with  potassium  and  sodium  is  usually  spoken 
of  as  indican,  but  should  not  be  confounded  with  the  vegetable  indi- 
can,  which,  as  has  been  shown,  is  a  glucoside  of  the  composition 
C26H31NO,7.  The  amount  which  is  daily  excreted  by  man  is  nor- 
mally small,  and  corresponds  to  about  0.0066  gramme.  Larger 
quantities  are  observed  when  from  any  cause  intestinal  putrefaction 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         251 

is  increased,  or  in  cases  in  which  putrefactive  changes  are  taking 
place  in  the  body  proper,  as  in  empyema,  providing  that  active 
resorption  can  occur. 

In  herbivorous  animals  larger  amounts  are  found  than  in  the 
carnivora.  Artificially  an  increased  elimination  can  be  effected  by 
feeding  animals  with  ortho-nitro-phenyl-propiolic  acid,  which  is 
reduced  in  the  body  to  indoxyl,  according  to  the  equation  : 

/C=COOH  /C.OH=CH 

C6H4;  +8H  =  C6H/  +3H20 

\no2  xnh 

Ortho-nitro-phenyl-  Indoxyl. 

propiolic  acid. 

Indican  crystallizes  in  colorless  platelets,  which  are  readily  soluble 
in  water  and  hot  alcohol,  while  in  cold  alcohol  they  dissolve  with 
great  difficulty.  On  decomposition  with  hydrochloric  acid  the 
indoxyl  is  obtained  in  the  form  of  oily  droplets  of  an  exceedingly 
offensive,  feculent  odor.  On  oxidation  this  is  then  transformed  into 
indigo-blue,  as  is  shown  in  the  equation  : 

/C.OH  =  CH  ,CO  v  /CO  N 

2C6H^  +20  =  C6H4<         >C=C<        >C6H4  +  2H20 

\NH  \nH/  xNH/ 

Indoxyl.  Indigo-blue. 

On  heating  an  aqueous  solution  of  indoxyl  to  a  temperature  of 
130°  C.  indoxyl-red  also  results.  This  is  a  brown  amorphous  sub- 
stance, which  is  insoluble  in  water,  but  dissolves  with  ease  in 
alcohol,  ether,  and  chloroform,  with  a  beautiful  red  color.  When 
Jaffe's  test  for  indican  is  applied  to  the  urine,  or  when  this  is  boiled 
and  treated  drop  by  drop  with  concentrated  nitric  acid  (Rosenbach's 
reaction),  a  mixture  of  a  blue  and  a  red  pigment  is  not  infrequently 
obtained,  and  it  is  quite  likely  that  the  latter  is  in  part  at  least  refer- 
able to  the  formation  of  the  indoxyl-red.  According  to  some  observ- 
ers, the  chromogen  of  this  substance  is  identical  with  the  so-called 
uroluematin,  and  the  pigment  is  probably  the  same  as  the  red  pig- 
ment of  Scherer,  the  urrhodin  of  Heller,  the  urorubin  of  Plosz,  the 
indirubin  of  Schunk,  the  indigo-purpurin  of  Bayer,  the  pigment 
of  Giacosa  and  others. 

The  blue  pigment  which  is  found  together  with  the  red  pigment 
when  urine  i-  treated  with  a  strong  mineral  acid  and  an  oxidizing 
agent  is,  :i-  lias  been  indicated,  indigo-blue,  and  is  identical  with 
urocyanin,  cyanurin,  Harnblau,  uroglaucin,  etc.,  of  former  observers. 
A-  ;i  general  rule,  its  amount  is  far  greater  than  that  of  the  red  pig- 
ment, and  is  at  times  the  only  one  that  is  obtained.  In  other  cases, 
however, the  red  seems  to  prevail, and  in  still  others  both  are  appar- 
ently present  in  aboul  equal  proportion.  The  cause  of  these  varia- 
tions i-  a-  vet  not  understood,  but  probably  rests  upon  variations  in 
bacterial  action  in  the  intestinal  tract.  As  a  general  rule,  indeed, 
notable  quantities  of  the  red  pigment  are  observed  only  under  patho- 
logical condition-. 


252  THE   URINE. 

Tests  for  Indican. — All  the  tests  employed  for  the  purpose  of 
demonstrating  the  presence  of  indican  in  the  urine  are  essentially 
based  upon  the  decomposition  of  the  substance,  with  the  liberation 
of  indoxyl  and  its  oxidation  to  indigo-blue. 

Jaffe's  Test,  as  modified  by  Stokvis. — A  few  cubic  cen- 
timeters of  urine  are  treated  with  an  equal  volume  of  concentrated 
hydrochloric  acid,  and  two  or  three  drops  of  a  strong  solution  of 
sodium  hypochlorite.  The  indigo-blue  which  thus  results  is  then 
extracted  by  shaking  with  a  little  chloroform.  If  red  pigment  has 
been  formed  at  the  same  time,  the  color  varies  from  a  violet  to  a 
purplish  red. 

If  it  is  desired  to  separate  the  two  pigments,  the  chloroform  extract 
is  evaporated  to  dryness  and  the  residue  washed  with  a  mixture  of 
equal  parts  of  96  per  cent,  alcohol,  ether,  and  water.  This  dissolves 
the  red  pigment  and  leaves  the  indigo-blue  behind.  Care  must  be 
had,  however,  not  to  add  too  much  of  the  hypochlorite  solution,  as 
otherwise  the  indigo-blue  is  oxidized  to  indigo-white,  and  no  color 
at  all  is  obtained.  Should  this  happen  after  the  addition  of  only 
one  or  two  drops,  the  following  test  had  better  be  employed,  as  a 
further  oxidation  is  here  not  effected  : 

Obeema  yer's  Test. — A  few  cubic  centimeters  of  urine  are  treated 
with  an  equal  volume  of  a  2  pro  mille  solution  of  ferric  chloride  in 
concentrated  hydrochloric  acid.  The  indigo-blue  is  extracted,  as 
above,  by  shaking  with  a  little  chloroform.  As  in  the  above  test, 
indoxyl-red  may  thus  also  be  obtained,  and  is  separated  from  the 
blue  pigment  as  just  described. 

Test  for  Urohaematin  (so-called). — A  small  amount  of  urine  is  thor- 
oughly agitated  with  chloroform  and  allowed  to  stand  for  a  few 
days.  The  chromogen  of  indoxyl-red  is  thus  extracted,  for  on  adding 
a  drop  of  concentrated  hydrochloric  acid  to  the  chloroform  extract 
a  beautiful  rose-color  appears,  which  varies  in  intensity  with  the 
amount  of  the  chromogen  present.  In  normal  urine  a  faint  reaction 
only  is  usually  seen ;  but  in  disease,  and  notably  in  ileus,  peritonitis, 
and  cancer  of  the  stomach,  I  have  repeatedly  met  with  more  indigo- 
red  than  indigo-blue. 

Rosenbach's  Reaction. — This  reaction  is  mostly  obtained  under 
pathological  conditions,  and  indicates  the  existence  of  greatly  in- 
creased intestinal  putrefaction.  It  is  referable  to  the  simultaneous 
formation  of  indigo-red  and  indigo-blue. 

While  boiling,  a  few  cubic  centimeters  of  urine  are  treated  drop 
by  drop  with  concentrated  nitric  acid  containing  a  little  nitrous 
acid,  when  in  the  presence  of  much  indigo-red,  viz.,  its  chromogen, 
the  urine  assumes  a  dark  Burgundy  color,  and  usually  shows  a 
bluish  tint  when  held  to  the  light.  On  standing,  the  red  pigment 
is  precipitated.  If  much  indigo-blue  is  present  at  the  same  time, 
as  is  usual,  the  foam  of  the  liquid  is  colored  blue.  On  adding  an 
excess  of  the  acid  the  color  often  disappears  and  the  urine  turns 
yellow. 


THE  AROMATIC  CONSTITUENTS  OF  THE    URINE.         253 

The  isolation  of  indican  from  urine  as  such  is  a  rather  complicated 
process,  and  need  not  be  described  at  this  place. 

Quantitative  Estimation. — Wang's  Method. — This  method  is 
based  upon  the  decomposition  of  the  indican  by  strong  hydrochloric 
acid,  and  the  ox  illation  of  the  resulting:  indoxyl  to  indigo-blue. 
This  is  then  transformed  into  indigo-sulphuric  acid,  and  estimated 
as  such  by  titration  with  a  solution  of  potassium  permanganate  of 
known  strength.  To  this  end,  a  stock  solution  of  the  salt  is  kept  on 
hand,  which  contains  about  3  grammes  to  the  liter.  Five  c.c.  of 
this  solution  are  diluted  with  195  c.c.  of  water,  when  the  titre  is 
ascertained  before  each  titration  by  comparing  it  with  a  dilute  solu- 
tion of  oxalic  acid.  The  amount  of  indigo-blue  which  each  cubic 
centimeter  represents  is  ascertained  by  multiplying  the  correspond- 
ing amount  of  oxalic  acid  by  1.04. 

The  amount  of  urine  which  is  necessary  varies  with  the  amount 
of  indican  present.  If  a  preliminary  test  gives  an  intense  reaction, 
from  25  to  50  c.c.  are  sufficient ;  otherwise  it  is  better  to  use  larger 
amounts,  as  from  200  to  250  c.c.  The  urine  is  then  precipitated 
with  a  20  per  cent,  solution  of  basic  lead  acetate,  care  being  taken 
to  avoid  an  excess.  A  large  portion  of  the  filtrate,  representing 
a  known  amount  of  urine,  is  then  treated  with  an  equal  volume 
of  Obermayer's  reagent,  and  extracted  with  chloroform  by  shaking. 
This  is  continued  with  portions  of  30  c.c.  until  the  chloroform  takes 
up  no  more  coloring-matter.  The  combined  extracts  are  freed  from 
chloroform  by  distillation.  The  residue  is  dried  for  a  few  minutes 
on  a  water-bath,  and  is  then  washed  with  a  mixture  of  equal  parts 
of  water,  ether,  and  alcohol  (96  per  cent.),  to  remove  the  reddish- 
brown  pigment  which  is  present  together  with  the  indigo-blue. 
The  solution  is  passed  through  a  small  filter,  so  as  to  collect  any 
particles  of  the  blue  pigment  which  may  be  present  in  suspension. 
The  filter  is  dried,  extracted  with  boiling  chloroform,  and  the 
resulting  solution  filtered  into  the  flask  containing  the  residual 
indigo-blue.  The  chloroform  is  distilled  off,  and  the  residue  treated 
with  3  or  4  c.c.  of  concentrated  sulphuric  acid,  while  still  warm. 
This  solution  is  allowed  to  stand  for  twenty-four  hours.  It  is  then 
poured  into  100  c.c.  of  water,  the  bottle  is  washed  out  with  a  little 
more  water,  when  the  solution  and  washings  are  filtered  and  titrated 
with  the  permanganate  solution.  The  color  at  first  changes  to 
green,  and   finally  becomes   yellowish  or  colorless.     The  calculation 

18  conducted  as  outlined   above. 

Skatoxyl  Sulphate. — Skatoxy]  sulphate,  like  indoxyl  sulphate, 
occurs  in  the  urine  in  combination  with  potassium  and  sodium.  Its 
amount,  however,  i<    normally  small,  and    it    may  at  times    be  absent 

altogether.  Larger  quantities  are  found  under  pathological  condi- 
tion-, associated  with  an  increased  degree  of  intestinal  putrefac- 
tion, and  it  may  then  happen  thai  more  skatoxy]  sulphate  is  found 
than  indican.  This,  however,  is  uncommon,  and  in  disease  also 
more   indican    i-   usually  present.     Like  indican,  it  is  decomposed 


254  THE   URINE. 

on  treating  with  concentrated  hydrochloric  acid,  and  on  subsequent 
oxidation  the  liberated  skatoxyl  yields  pigments  which  are  for  the 
most  part  of  a  red  color.  Of  their  chemical  nature,  however, 
nothing  is  known.  One  of  these  may  possibly  be  identical  with 
Rosin's  urorosein.  Rosin,  to  be  sure,  claims  that  the  chromogen  of 
urorosein  is  not  a  conjugate  sulphate  but  we  know  that  a  portion  of 
the  skatol  also  appears  in  combination  with  glucuronic  acid  in  the 
urine,  and  it  is  hence  possible  that  his  pigment  may  be  derived  from 
this  source.  Urines  containing  notable  quantities  of  skatoxyl 
become  darker  on  exposure  to  the  air,  and  may  gradually  turn  a 
reddish-violet  or  almost  a  black  color.  This  change,  as  in  the 
phenol  urines,  begins  at  the  surface  and  gradually  extends  down- 
ward. 

Tests. — To  demonstrate  the  presence  of  skatoxyl  in  urine,  this 
is  strongly  acidified  with  hydrochloric  acid  and  extracted  with 
amyl  alcohol,  which  takes  up  the  coloring-matter.  Chloroform 
and  ether  do  not  dissolve  this  in  acid  solution,  but  do  so  in  neutral 
or  alkaline  solution,  providing  that  the  pigment  has  been  freshly 
formed. 

Test  for  Urorosein  (so-called). — A  few  cubic  centimeters  of  urine 
are  treated  with  an  equal  amount  of  concentrated  hydrochloric  acid 
and  one  or  two  drops  of  a  strong  solution  of  calcium  hypochlorite. 
The  indigo-blue  is  extracted  with  chloroform,  and  it  will  now  be 
observed  that  the  supernatant  fluid  presents  a  red  color.  On  shaking 
with  amyl  alcohol  this  takes  up  the  red  pigment,  which  is  thus 
manifestly  not  identical  with  indigo-red.  Upon  the  addition  of 
sodium  hydrate  to  the  alcoholic  solution  the  color  disappears,  but 
reappears  upon  the  subsequent  addition  of  hydrochloric  acid.  On 
standing,  the  color  gradually  disappears.  Under  normal  conditions 
this  reaction  is  not  well  marked,  but  becomes  quite  distinct  in  cases 
in  which  intestinal  putrefaction  is  much  increased. 

A  portion  of  the  skatol,  as  I  have  already  stated,  appears  in  the 
urine  as  skatol-carbonic  acid: 

/C(CH3k 
QH4<  >C.COOH. 

The  substance  is  usually  present  in  exceedingly  small  amounts, 
however,  and  has  not  been  isolated  in  substance.  To  demonstrate 
its  presence  even,  it  is  necessary  to  work  with  several  liters  of 
urine.  To  this  end,  the  fluid  (5000-6000  c.c.)  is  evaporated  to  a 
syrup ;  the  residue  is  extracted  with  absolute  alcohol,  the  alcoholic 
solution  is  evaporated,  and  the  remaining  material  extracted  with 
ether  after  acidifying  strongly  with  sulphuric  acid.  From  the 
ethereal  solution  the  substance  is  obtained  in  aqueous  solution  by 
shaking  with  a  dilute  solution  of  sodium  hydrate.  The  alkaline 
solution  is  then  evaporated  and  the  residue  extracted  with  alcohol. 
This  extract  is  now  concentrated  to  about  100  c.c.  and  precipitated 


THE  AROMATIC  CONSTITUENTS  OF  THE   URINE.         255 

with  an  equal  volume  of  ether.  The  filtrate  is  evaporated  to  dry- 
ness, the  residue  acidified  with  hydrochloric  acid,  and  extracted  with 
ether.  The  residue  of  the  ethereal  extract  is  then  finally  dissolved 
in  hot  water.  To  remove  the  remaining  hydrochloric  acid,  this 
solution,  after  filtering  and  cooling,  is  again  evaporated  to  dryness 
and  redissolved  in  hot  water.  To  demonstrate  the  presence  of 
skatol-carbonic  acid,  a  portion  of  this  final  solution  is  then  treated 
with  a  few  drops  of  pure  nitric  acid  and  a  trace  of  potassium 
nitrite.  A  red  color  thus  develops,  which  may  be  extracted  with 
amvl  alcohol  or  acetic  ether.  This  solution  shows  a  band  of  absorp- 
tion in  the  green  portion  of  the  spectrum.  On  adding  a  solution  of 
sodium  hydrate  to  the  ethereal  solution  the  red  color  disappears,  but 
reappears  on  the  subsequent  addition  of  hydrochloric  acid. 

In  addition  to  the  aromatic  bodies  which  have  thus  far  been  con- 
sidered, traces  of  the  aromatic  oxy-acids  may  also  appear  in  the 
urine  in  combination  with  sulphuric  acid,  but  the  amount  is  exceed- 
ingly small,  and  may  well  be  ignored. 

The  Conjugate  Glucuronates. 

Glucuronic  acid  as  such  does  not  occur  in  the  urine.  The  sub- 
stance can  combine  with  various  aromatic  bodies,  however,  and  may 
in  this  manner  escape  further  oxidation  in  the  body.  Normally,  it 
is  found  only  in  traces,  in  combination  with  indoxyl,  skatoxyl, 
phenol,  and  paracresol,  while  the  greater  portion  of  these  substances 
is  eliminated  in  the  form  of  conjugate  sulphates,  as  has  been  pointed 
out.  Larger  quantities  are  found  in  the  urine  after  the  administra- 
tion of  chloral,  camphor,  naphtol,  oil  of  turpentine,  menthol,  toluol, 
euxanthin,  morphin,  etc.  With  these  it  forms  compounds  which  are 
closely  related  to  the  glucosides.  According  to  the  character  of  the 
aromatic  component,  the  resulting  glucuronates  have  been  termed 
campho-glucuronic  acid,  menthol-glucuronic  acid,  urochloralic  acid, 
phenyl-indoxyl-skatoxyl-glucuronic  acid,  etc. 

Of  the  origin  of  the  glucuronic  acid,  and  its  fate  under  normal 
conditions,  we  know  little.  That  it  is  formed  in  the  tissues  of  the 
body  is  apparent  from  the  fact  that  even  in  a  starving  animal  the 
administration  of  camphor,  chloral,  etc.,  leads  to  elimination  of  these 
substances  in  combination  with  the  acid  in  question.  As  glucuronic 
acid  is  a  derivative  of  glucose,  we  may  thus  imagine  that  during 
starvation  it  is  derived  from  glycogen,  or  even  from  the  albumins, 
through  a  splitting  off  of  the  carbohydrate  group.  \t  has  been 
demonstrated,  a-  a  matter  of  fact,  that  the  formation  of  glycogen  in 
the  liver  can  be  artificially  increased  by  introducing  glucuronic  acid 
with  the  food.  The  chemical  relation  of  glucuronic  acid  to  glucose 
ha-  already  been  considered.  On  oxidation  glucose  thus  first  yields 
the  monobasic  glucuronic  acid, and  then  the  dibasic  saccharinic  acid. 
The  latter  in  turn  may  be  transformed  into  saccharo-lactonic  acid, 
which  on  reduction  yields  glucuronic  acid,  so  that  this  stands  mid- 


256  THE    URINE. 

way  between  gluconic  acid  and  saccharinic  acid.  These  relations 
are  shown  by  the  formulas  : 

CH2.OH  —  (CH.OH)4  —  COH,  glucose. 

CH2.OH  —  (CH.OH)4  —  COOH,  gluconic  acid. 

COOH     —  (CH.OH)4  —  COH,  glucuronic  acid. 

COOH.    —  (CH.OH)4  —  COOH,  saccharinic  acid. 

On  boiling  with  water  glucuronic  acid  is,  in  part  at  least,  trans- 
formed into  its  anhydride,  glucuron,  C6H806. 

Aside  from  its  probable  origin  from  glycogen,  glucuronic  acid  may 
also  be  derived  from  chondroitin-sulphuric  acid,  which  has  also  been 
observed  in  normal  urine.  We  have  seen,  as  a  matter  of  fact,  that 
this  acid  occurs  in  the  cartilaginous  structures  of  the  higher  animals, 
and  that  its  hyalin  component  chondroitin  first  yields  chondrosin  on 
hydrolytic  decomposition,  which  is  then  further  decomposed  into 
glucuronic  acid  and  glucosamin,  as  shown  in  the  equations  : 

(1)  C18H27NOu  +  3H20  =  C12H21NOu  +  3CH3.COOH 
Chondroitin.                         Chondrosin.  Acetic  acid. 

(2)  C12H21NO„  +  H20    =  COOH.(CHOH)4.COH  +  C6Hn06.NH2 
Chondrosin.  Glucuronic  acid.  Glucosamin. 

Schmiedeberg,  indeed,  regards  the  chrondroitin-sulphuric  acid  as  the 
normal  source  of  the  glucuronic  acid. 

Glucuronic  acid  has  thus  far  not  been  obtained  in  crystalline 
form.  It  is  a  syrupy  substance  which  is  readily  soluble  in  water 
and  alcohol.  Its  anhydride,  however,  is  a  crystalline  body,  and  is 
likewise  soluble  in  water  but  insoluble  in  alcohol.  The  free  acid 
and  its  alkaline  salts  are  dextrorotatory,  while  the  conjugate  glucuro- 
nates  turn  the  polarized  light  to  the  left.  The  free  acid,  moreover, 
as  well  as  its  salts  and  most  of  its  compound  ethers,  reduces  the 
oxides  of  copper,  bismuth,  and  silver  in  alkaline  solution,  and  it  is 
thus  possible  to  confound  them  with  glucose  if  reliance  is  placed  upon 
the  corresponding  tests  alone.  "With  phenyl-byclrazin  the  free  acid 
is  said  to  form  a  crystalline  compound  with  a  melting-point  of  114°— 
115°  C.  Unlike  glucose,  it  is  non-fermentable.  It  gives  the  fur- 
furol  reaction  and  simulates  the  pentoses  in  reacting  with  phloro- 
glucin  hydrochlorate,  but  not  with  orcin  (see  page  284). 

To  demonstrate  the  presence  of  glucuronic  acid  in  the  urine,  it 
is  necessary  to  isolate  its  compound  ethers  and  to  decompose  these 
with  superheated  steam.  Under  normal  conditions  this  is  practi- 
cally impossible  unless  very  large  quantities  of  urine  are  employed. 
Its  presence  may,  however,  be  suspected  if  a  urine  reduces  Fehling's 
and  Nylander's  solution,  if  it  is  lsevorotatory,  and  gives  the  phloro- 
glucin  hydrochlorate  reaction,  while  sugar  is  absent. 

The  Compound  Glycocolls. 

As  has  been  pointed  out,  phenyl-propionic  acid  and  phenyl-acetic 
acid,  which  are   both  formed  from  albuminous  material  during  the 


THE  AROMATIC  CONSTITUENTS  OF  THE    URINE.  257 

process  of  intestinal  putrefaction,  are  in  part  absorbed  in  the  intestinal 
tract,  and  are  eliminated  in  the  urine  in  combination  with  glycocoll 
as  hippuric  acid  and  phenaceturic  acid,  respectively.  But  while 
phenvl-acetic  acid  unites  with  glycocoll  directly,  phenyl-propionic 
acid  is  usually  first  oxidized  to  benzoic  acid  (see  page  97). 

Hippuric  Acid. — While  a  certain  amount  of  the  benzoic  acid 
which  enters  into  the  construction  of  the  hippuric  acid  molecule  is 
derived  from  the  phenyl-propionic  acid  which  results  during  the 
process  of  intestinal  putrefaction,  another  portion  is  ingested  as 
such,  or  in  the  form  of  other  aromatic  substances  which  can  be 
transformed  into  benzoic  acid  in  the  animal  body.  We  can  thus 
understand  why  larger  amounts  of  hippuric  acid  are  encountered 
in  the  urine  of  herbivorous  animals  than  in  that  of  the  carnivora,  as 
the  food  of  the  former  always  contains  very  considerable  amounts  of 
toluol,  cinnamic  acid,  quinic  acid,  etc.,  all  of  which  may  give  rise  to 
benzoic  acid.  In  man  the  daily  elimination  corresponds  to  about 
0.7  gramme,  but  may  be  increased  by  the  ingestion  of  such  articles 
of  food  as  cranberries,  primes,  reine-claudes,  etc. 

In  a  few  instances  the  substance  has  been  found  in  urinary 
sediments. 

While  the  source  of  the  aromatic  component  of  hippuric  acid 
is  thus  quite  well  understood,  we  know  but  little  of  the  origin  of 
glycocoll.  To  a  certain  extent  this  may  also  be  formed  during  the 
process  of  albuminous  putrefaction,  as  we  know  that  all  albumins 
contain  a  glycocoll  radicle,  but  there  is  reason  to  believe  that  it 
may  likewise  originate  within  the  tissues  of  the  body.  It  mani- 
festly plays  an  important  role  in  the  process  of  metabolism,  for  we 
have  seen  that  to  a  certain  extent,  no  doubt,  it  is  concerned  in  the 
formation  of  urea,  and  it  has  also  been  noted  that  in  man  and  in 
other  animals  a  very  considerable  proportion  of  cholalic  acid  is  elimi- 
nated  from  the  body  in  combination  with  glycocoll. 

Through  the  interesting  researches  of  Sehmiedeberg  we  know  that 
the  synthesis  of  glycocoll  and  benzoic  acid  is  in  dogs  effected  in  the 
kidneys  exclusively,  in  other  animals,  however,  this  process  may 
occur  in  other  organs  as  well,  for  it  has  been  shown  that  after 
removal  of  the  kidneys  hippuric  acid  can  be  isolated  from  the  liver 
and  the  muscles,  at  least,  if  benzoic  acid  has  been  previously 
administered. 

Properties. — Hippuric  acid  crystallizes  in  long  rhombic  prisms 
when  allowed  to  separate  from  its  solutions  slowly,  while  when 
rapidly  formed,  and  especially  if  the  amount  is  small,  it  occurs  in 
long  nee<lle~  which  are  frequently  grouped  in  star"  *>nd  rosettes. 
The  melting-point  of  the  substance  is  1*7. o  ( !.  It  is  i  table  with 
great  difficulty  in  cold  water  and  ether,  while  in  hot  water  and 
alcohol  it  dissolves  with  comparative  ease.     In  aqueous  solutions  of 

the  alkaline  hydrates  and    carbonates  it  dissolves  with  the   formation 
of  the   corresponding   -:ilt-,   from  which    the    free  acid    may  again    be 
obtained  by  acidifying  with  a  mineral  acid. 
17 


258  THE   URINE. 

On  boiling  with  dilute  mineral  acids  or  alkalies  hippuric  acid  is 
decomposed  into  its  components.  The  same  result  is  reached  if 
ammoniacal  decomposition  is  allowed  to  occur,  and  in  such  urines 
benzoic  acid  only  is  found.  In  traces,  benzoic  acid  is  said  to  occur 
in  every  urine  together  with  hippuric  acid,  and  it  is  thought  that  its 
presence  under  normal  conditions  may  be  due  to  the  action  of  a 
ferment,  the  so-called  histenzyme  of  Schmiedeberg,  which  has  been 
found  in  the  kidneys,  and  which  is  known  to  be  capable  of  effecting 
the  decomposition  of  hippuric  acid  as  outlined. 

On  heating  hippuric  acid  in  a  dry  test-tube  it  melts,  and  is  then 
decomposed  with  the  formation  of  benzoic  acid,  which  sublimes  in 
the  upper  portion  of  the  tube.  The  liquid  mass  at  the  same  time 
assumes  a  red  color,  and  develops  an  odor  which  at  first  is  suggestive 
of  hay,  but  subsequently  resembles  that  of  hydrocyanic  acid.  This 
reaction,  together  with  the  form  of  the  crystals  and  their  insolubility 
in  petroleum-ether,  serves  to  distinguish  hippuric  acid  from  benzoic 
acid.  But  like  this,  it  develops  a  marked  odor  of  bitter  almonds 
when  it  is  evaporated  with  nitric  acid  and  the  residue  is  then 
heated.  The  reaction  is  due  to  the  formation  of  nitrobenzol. 
In  the  urine  hippuric  acid  is,  of  course,  not  present  in  the  free 
state,  but  in  combination  with  alkalies,  and  notably  potassium  and 
sodium. 

Synthesis  of  Hippuric  Acid. — Hippuric  acid  can  be  formed  syn- 
thetically in  vitro  also  from  benzoic  acid  and  glycocoll  by  heating 
the  two  substances  together  at  a  temperature  of  160°  C.  in  a  sealed 
tube.  In  a  similar  manner  it  is  obtained  from  benzamide  and 
monochlor-acetic  acid.  The  reactions  which  take  place  may  be 
represented  by  the  equations  : 

C6H5.COOH     +  CH2(NH2).COOH  =  CH2.NH(C6H5.CO).COOH  +  H20 
Benzoic  Glycocoll.  Hippuric  acid, 

acid. 

C6H5.CO.NH2  +  CH,.Cl.COOH        =  CH2.NH(C6H5.CO).COOH  +  HC1 
Benzamide.  Monochlor-  Hippuric  acid. 

acetic 
acid. 

Isolation  of  Hippuric  Acid. — Hippuric  acid  is  most  conveniently 
isolated  from  the  urine  of  herbivorous  animals,  in  which,  as  has 
been  stated,  it  is  present  in  much  greater  amounts  than  in  that  of 
man  and  the  carnivorous  animals.  To  this  end,  1000  c.c.  of  fresh 
urine  are  rendered  feebly  alkaline  with  milk  of  lime  and  boiled  for  a 
few  minutes.  The  liquid  is  filtered  while  still  warm,  concentrated, 
and  on  cooling  acidified  with  hydrochloric  acid  added  in  moderate 
excess.  After  twenty-four  hours  the  crystals  of  hippuric  acid  which 
have  separated  out  are  filtered  off,  and  are  freed  from  adhering  pig- 
ments as  follows  :  they  are  dissolved  in  hot  water,  and  treated  with 
alum  and  then  with  an  amount  of  sodium  carbonate  sufficient  to 
cause  the  formation  of  an  abundant  precipitate ;  the  reaction,  how- 
ever, should  remain  still  acid.  The  pigments  are  retained  by  the 
aluminous  precipitate.     The  filtrate  is  then  strongly  concentrated, 


THE  AROMATIC  CONSTITUENTS   OF  THE   URINE.         259 

acidified  with  hydrochloric  acid,  and  allowed  to  stand.  In  this 
manner  the  hippuric  acid  is  obtained  in  fairly  pure  form,  and  can  be 
further  purified,  if  desired,  by  recrystallization  from  hot  water. 

Isolation  of  Glycocoll  and  Benzoic  Acid  from  Hippuric  Acid. — As 
stated  before,  glycocoll  is  most  conveniently  obtained  from  hippuric 
arid.  To  effect  the  decomposition,  this  is  boiled  for  ten  to  twelve 
hours  with  four  parts  of  diluted  sulphuric  acid.  On  cooling-,  the 
benzoic  acid  which  has  separated  out  is  filtered  off.  The  filtrate  is 
concentrated  and  extracted  with  ether,  which  takes  up  the  remain- 
ing benzoic  acid.  The  sulphuric  acid  is  now  removed  by  means  of 
barium  hydrate  and  the  excess  of  barium  precipitated  with  carbon 
dioxide.  The  filtrate  is  concentrated,  when  on  cooling  the  glycocoll 
is  obtained  in  crystalline  form. 

Isolation  and  Quantitative  Estimation. — Five  hundred  to  one  thou- 
sand c.c.  of  urine  are  rendered  slightly  alkaline  with  sodium  car- 
bonate, and  are  evaporated  to  a  thick  syrup,  taking  care  that  the 
reaction  remains  alkaline.  It  is  then  extracted  with  cold  strong 
alcohol  (90  to  95  per  cent.)  by  adding  about  one-half  the  volume  of 
the  original  solution,  and  allowing  the  mixture  to  stand  for  twenty- 
four  hours.  It  is  then  filtered  and  the  alcohol  distilled  off.  The 
remaining  aqueous  solution  is  acidified  with  dilute  sulphuric  acid, 
and  the  liberated  hippuric  acid  extracted  with  several  portions  of 
acetic  ether  The  ethereal  solution  is  evaporated  to  dryness,  when 
the  remaining  impurities,  such  as  phenols,  aromatic  oxy-acids, 
benzoic  acid,  and  fat,  are  removed  by  washing  with  cold  petroleum- 
ether,  in  which  hippuric  acid  is  insoluble.  It  is  then  dissolved  in 
warm  water,  and  the  solution  evaporated  at  a  temperature  of  from 
50°  to  60°  C.  until  crystallization  occurs.  On  cooling,  the  crystals 
are  filtered  off  and  weighed.  The  mother-liquor  is  extracted  with 
acetic  ether,  the  ethereal  solution  is  evaporated  to  dryness,  and  the 
weight  of  the  residue  added  to  that  of  the  crystals. 

Phenaceturic  Acid. — In  small  amounts  phenaceturic  acid  has 
been  repeatedly  obtained  from  the  urine  of  man,  but  is  principally 
met  with  in  that  of  herbivorous  animals,  in  which  the  putrefactive 
processes,  owing  to  the  greater  length  of  the  intestinal  tract  and  the 
character  <>('  the  food,  are  more  extensive.  On  boiling  with  dilute 
mineral  acids  it  is  decomposed  into  its  components,  as  shown  in  the 
equation  : 

<  :i    Nil  <  II  .<    M  ,<<>,  .COOH       H,o      rn,(Cfiir-,).n>(>ir   |   CH2(NHaVCOOH 
Phenaceturic  acid.  Phenyl-acetic  acid.  Glj  cocoll. 

Properties. —  Phenaceturic  acid  crystallizes  in  small  rhombicqdatos 
with  rounded  angles,  which  are  very  similar  to  the  corresponding 
crystals  of  uric  acid. 

Isolation. — Phenaceturic  acid  may  be  isolated  from  the  urine  of 
the  horse  after  separation  of  the  hippuric  acid,  as  shown  above. 
The  mother-liquor  i-  then  extracted  with  acetic  ether,  which  takes 
id  th<-  acid.     On  evaporation  the  residue  is  dissolved  in  a  dilute 


260  THE   URINE. 

solution  of  sodium  hydrate.  From  this  solution  the  substance  is 
again  extracted  with  ether,  after  acidifying  with  hydrochloric  acid, 
and  thus  purified,  and  finally  allowed  to  crystallize  from  its  aqueous 
solution. 

In  this  connection  brief  reference  may  be  made  to  omithuric  acid, 
which  may  be  obtained  from  the  urine  of  birds  when  these  are  fed 
with  benzoic  acid,  and  which  probably  also  represents  a  normal  con- 
stituent of  such  urine.  Its  formation  is  analogous  to  that  of  hip- 
puric  acid  in  mammals,  but  the  benzoic  acid  here  combines  with  a 
diamido-acid,  ornithin,  which  is  a,  o-diamido-valerianic  acid,  as  has 
been  shown  before. 

The  relation  of  ornithin  to  arginin  has  already  been  considered. 

THE   AROMATIC   OXY-ACIDS. 

The  aromatic  oxy-acids  which  may  be  found  in  the  urine  under 
normal  conditions  are  para-oxy phenyl-propionic  acid  or  hydro- 
paracumaric  acid,  and  para-oxyphenyl-acetic  acid,  which  results 
from  the  former  on  oxidation.  Both  are  derivatives  of  tyrosin, 
and  are  formed  during  the  process  of  intestinal  putrefaction,  as 
has  been  shown.  Para-oxy phenyl-lactic  acid  may  further  be 
encountered  when  tyrosin  has  been  administered  to  animals  in 
large  amounts.  Under  pathological  conditions,  as  in  acute  yellow 
atrophy,  where  leucm  and  tyrosin  may  appear  in  the  urine  as  such, 
para-oxyphenyl-glycolic  acid  or  oxy-amygdalic  acid  has  also  been 
found.  Of  special  interest  finally  is  the  fact  that  in  some  instances 
dioxyphenyl-acetic  acid  (homogentisinic  acid)  and  trioxyphenyl-pro- 
pionic  acid  or  uroleucinic  acid  also  may  occur  in  the  urine.  The 
chemical  relationship  which  exists  between  these  various  substances 
and  tyrosin,  from  which  they  are  all  probably  derived,  is  apparent 
from  their  formula? : 


CfiH 


6-LJ-4 


C6H4< 


C  H 


/OH(l) 
CH2.CH(NH).COOH(4)   para-oxyphenyl-amido-propionic  acid  (tyrosin). 

X>H(1) 

vCH2.CH2.COOH  para-oxyphenyl-propionic  acid. 

,(OH)3 

vCH2.CH2.COOH  trioxyphenyl-propionic  acid. 


C  H 

'XCH2.CH(OH).COOH  para-oxyphenyl-lactic  acid. 

.OH 

C6H4< 

Nm^COOH  para-oxyphenyl-acetic  acid. 


THE  AROMATIC  OXY- ACIDS.  261 

/(OH), 

xCH,.COOH  dioxyphenyl-acetic  acid. 

.OH 

c«hZ 

CH(OHj.COOH        para-o.xyphenyl-glycolic  acid. 

As  has  been  pointed  out,  the  two  aromatic  oxy-acids  which  are 
usually  found  in  the  urine  originate  in  the  intestinal  tract  during 
the  process  of  albuminous  putrefaction.  There  is  reason  to  believe, 
moreover,  that  homogentisinic  acid  and  uroleucinic  acid  are  likewise 
of  intestinal  origin,  and  are  referable  to  the  activity  of  a  special 
micro-organism,  which  is  usually  not  found.  But  as  para-oxy- 
phenyl-glyeolic  acid  manifestly  originates  in  the  tissues  of  the  body, 
we  must  admit  that  the  other  members  of  the  group  may  also  be 
formed  beyond  the  intestinal  tract.  To  what  extent  this  occurs, 
however,  we  do  not  know. 

_  A  certain  fraction  of  these  bodies  appears  in  the  urine  in  com- 
bination with  sulphuric  acid,  but  the  greater  portion  is  eliminated 
as  such,  viz.,  as  potassium  and  sodium  salts.  The  amount  of  the 
common  oxy-acids,  however,  is  always  small,  and  rarely  exceeds  0.03 
gramme  in  the  twenty-four  hours. 

To  demonstrate  the  presence  of  the  common  oxy-acids  in  the 
urine,  we  proceed  as  follows  :  500  c.c.  of  urine  are  strongly  acidified 
with  hydrochloric  acid  and  distilled  until  the  phenols,  viz.,  phenol 
and  paracresol,  have  passed  over.  This  can  be  recognized  by  test- 
ing the  distillate  from  time  to  time  with  Millon's  reagent,  On 
cooling,  the  remaining  fluid  is  thoroughly  extracted  with  ether, 
which  takes  up  the  oxy-acids  as  well  as  pyrocatechin  and  hydro- 
quinon.  To  separate  the  acids  from  the  latter,  the  ethereal  extract 
is  shaken  with  a  dilute  solution  of  sodium  carbonate.  The  acids  are 
thus  transformed  into  the  corresponding  salts  and  are  found  in  the 
alkaline  solution.  After  separation  from  the  ether  this  is  acidified 
with  dilute  sulphuric  acid  and  extracted  with  ether.  The  ethereal 
solution  contain-;  the  free  oxy-acids.  Their  presence  can  be  demon- 
strated by  evaporating  to  dryness,  when  the  residue  is  dissolved  in  a 
little  water  and  tested  with  Millon's  reagent. 

To  isolate  the  individual  acid-,  much  larger  Quantities  of  urine 
are  necessary.  We  may  then  proceed  as  described  in  the  section  on 
ih-  Feces. 

Homogentisinic  Acid.— The  presence  of  homogentisinic  acid 
may  he  suspected  if  a  urine,  on  being  rendered  alkaline,  turns  a 
dark  reddish-brown  on  standing,  and  ultimately  becomes  black.  At 
the  same  time  a  positive  reaction  with  Fehlingpa  solution  is  obtained, 
while  polar i metric  examination  shows  that  the  urine  is  optically 
inactive.  \vland< ■!■'-  solution  is  not  reduced.  Upon  the  addition 
of  a  small  amount  of  a  dilute  solution  of  ferric  chloride  a  greenish- 
blue  color  develop-,  which  is  only  of  momentary  occurrence,  how- 
ever. 


262  THE   URINE. 

Boedeker  was  the  first  to  describe  a  urine  of  this  kind,  and  termed 
the  substance  giving  rise  to  the  above  reaction  alkapton.  Subse- 
quently, however,  he  expressed  the  opinion  that  his  alkapton  may 
have  been  pyrocatechin.  Other  investigators  have  isolated  sub- 
stances from  such  urines,  which  have  been  variously  termed  pyro- 
catechuic  acid,  urrhodinic  acid,  glycosuric  acid,  uroxanthinic  acid, 
and  uroleucinic  acid,  but  there  is  reason  to  suppose  that,  with  the 
possible  exception  of  the  last  mentioned,  all  these  substances  are 
identical  with  homogentisinic  acid.  This  was  first  isolated  from  an 
"  alkapton  "  urine  by  Baumann  and  Wolkow,  and  has  since  been 
found  in  every  case  that  has  been  examined  in  this  direction. 

Alkaptonuria,  though  it  may  occur  in  disease,  is  generally  regarded 
as  the  expression  of  an  unusual  form  of  intestinal  putrefaction 
which  in  no  way  affects  the  health  of  the  individual.  Some  ob- 
servers, on  the  other  hand,  look  upon  it  as  a  metabolic  abnormality, 
and  it  must  be  confessed  that  micro-organisms  have  thus  far  not 
been  isolated  from  the  intestinal  contents  of  such  cases  which  are 
capable  of  effecting  the  transformation  of  tyrosin  to  homogentisinic 
acid  in  vitro.  That  tyrosin,  indeed,  is  its  ultimate  source  cannot  be 
doubted,  and  it  has  been  shown,  as  a  matter  of  fact,  that  following 
the  administration  of  this  substance  homogentisinic  acid  appears  in 
the  urine  in  greatly  increased  amount.  Baumann  thus  noted  that 
while  the  average  elimination  in  one  of  his  cases  amounted  to  4.6 
grammes,  14  grammes  were  once  extracted  in  the  twenty-four  hours 
after  tyrosin  had  been  ingested. 

Isolation. — Homogentisinic  acid  may  be  conveniently  isolated 
from  the  urine  according  to  the  method  suggested  by  Garrod.  The 
collected  urine  of  twenty-four  hours  is  heated  nearly  to  the  boiling- 
point,  and  then  treated  with  5  or  6  grammes  of  neutral  lead  acetate 
in  substance  for  every  100  c.c.  of  the  urine.  As  soon  as  the  salt  is 
dissolved,  the  resulting  precipitate  is  filtered  off  and  the  filtrate  set 
aside  in  the  cold  for  twenty-four  hours.  The  crystals  of  lead  homo- 
gentisinate  are  then  collected  on  a  filter  and  dissolved  in  hot  water. 
This  solution  gives  the  various  reactions  described  above.  To  isolate 
the  free  acid,  the  lead  compound  is  decomposed  with  hydrogen  sul- 
phide and  the  filtrate  carefully  evaporated  on  a  water-bath  until  the. 
fluid  begins  to  darken,  when  it  is  further  concentrated  in  a  vacuum 
to  the  point  of  crystallization.  The  resulting  crystals  are  soluble  in 
water,  alcohol,  and  ether,  but  are  insoluble  in  chloroform,  benzol, 
and  toluol.     They  melt  at  146.5°-147°  C. 

Uroleucinic  acid  has  been  found  only  once  in  an  alkapton  urine. 
In  its  general  reactions  it  resembles  homogentisinic  acid,  but  does  not 
give  the  iron  reaction  described  above.  Unlike  the  latter,  it  reduces 
Nylander's  solution  when  present  to  the  extent  of  0.5  per  cent,  or 
more. 

Inosit. — The  origin  and  chemical  constitution  of  inosit  will  be 
considered  elsewhere.  According  to  Hoppe-Seyler,  it  may  occur  in 
the  urine  under  normal  conditions,  but  more  commonly  it  is  found 


THE  AROMATIC  OXY- ACIDS.  263 

in  diseases  which  are  associated  with  a  high  grade  of  polyuria,  such 
as  diabetes  insipidus,  diabetes  mellitus,  in  chronic  interstitial  neph- 
ritis, etc. 

To  demonstrate  its  presence  in  the  urine  large  amounts  are  con- 
centrated to  a  syrup,  which  is  then  extracted  with  four  times  its 
volume  of  alcohol  by  boiling.  On  cooling,  this  extract  is  treated 
with  an  excess  of  ether,  when  the  inosit  gradually  crystallizes  out 
and  may  be  recognized  by  its  special  tests  (see  page  360). 

The  remaining  aromatic  substances  which  have  been  found  in  the 
urine  are  the  kynurenic  acid  and  urocaninic  acid  of  the  urine  of 
dogs,  and  the  so-called  lithuric  acid,  which  has  been  obtained  from 
the  urine  of  the  ox.  The  two  latter  have  thus  far  been  found  in 
only  one  instance  and  need  not(  be  considered  at  this  place.  Their 
formulae  are  given  as  : 

CjjH^X/)^  urocaninic  acid. 

C15H,9X09,  lithuric  acid. 

The  so-called  damalic  acid  and  damaluric  acid,  which  are  obtained 
from  the  urine  of  the  horse  and  the  cow,  probably  represent  a  mixt- 
ure of  benzoic  acid  and  volatile  fatty  acids. 

Kynurenic  Acid. — Kynurenic  acid  is  said  to  be  a  constant  con- 
stituent of  the  urine  of  dogs,  and  is  supposedly  formed  during  the 
process  of  intestinal  putrefaction.  Its  mother-snbstance  is  mani- 
festly an  albuminous  derivative,  as  the  amount  which  appears  in  the 
urine  is  largely  dependent  upon  the  quantity  of  albuminous  food 
ingested.  Of  its  chemical  nature  and  immediate  antecedents,  how- 
ever, nothing  is  known. 

Kynurenic  acid  is  now  regarded  as  oxyquinolin-carbonic  acid,  and 
is  decomposed  by  heat,  with  the  formation  of  carbon  dioxide  and  a 
basic  substance,  kynurin.  On  reduction  the  latter  is  transformed 
into  quinolin.     These  changes  are  represented  by  the  equations : 

(1)  CH.C9H5N.COOH  =  C9IIftNO  +  C02. 

Kynurenic  acid.  Kynurin. 

(2)  C9II7NO    I    _>II       =C9HTN     +H20. 
Kynurin.  Quinolin. 

Isolation. — To  isolate  kynurenic  acid  from  the  urine,  500  c.c.  are 
treated  with  hydrochloric  acid  in  the  proportion  of  4  c.c.  for  every 
loo  p.c.  of  lb''  urine.  On  standing  for  forty-eight  hours  the  sub- 
stance  in  question  crystallizes  out  together  with  uric  acid.  To  sepa- 
rate it  from  tin-  latter,  dilute  ammonia  i~  added  to  the  crystalline 
precipitate.  The  uric  acid  remains,  and  from  its  ammoniacal  solu- 
tion the  kynurenic  acid  is  then  precipitated  by  acidifying  with  hydro- 
chloric acid. 

The  crystals  are  soluble  in  alcohol  mid  melt  at  253°  C.  On 
porating  a  bit  of  the  materia]  with  hydrochloric  acid  and  potas- 
sium  chlorate  on  a  porcelain   plate  a   reddish  residue  is  obtained, 


264  THE   URINE. 

which  principally  consists  of  tetrachloro-oxykynurin.  When  this 
is  moistened  with  ammonia  a  brownish-green  color  develops,  and  on 
standing  this  soon  passes  into  a  fine  emerald  green. 

The  Fatty  Acids. 

The  Volatile  Fatty  Acids. — The  volatile  fatty  acids  which  may 
be  isolated  from  any  urine,  and  which  are  especially  abundant  in 
that  of  herbivorous  animals,  are  normally  derived  from  the  intestinal 
tract,  where  they  are  principally  formed  during  the  process  of  carbo- 
hydrate fermentation  ;  a  certain  fraction,  however,  is  also  referable 
to  albuminous  putrefaction.  These  acids  are  acetic  acid,  formic  acid, 
propionic  acid,  and  butyric  acid. 

The  non-volatile  acids,  capric  acid  and  caprylic  acid,  have  further 
been  found  in  the  urine  of  herbivorous  animals. 

In  man  about  0.05  gramme  of  volatile  fatty  acids  is  excreted  in 
twenty-four  hours.  Especially  large  amounts,  such  as  3  grammes  pro 
die,  have  been  found  in  the  urine  of  the  goat.  In  various  diseases 
larger  amounts  have  also  been  encountered  in  man,  but  it  is  still  an 
open  question  whether  they  are  then  derived  from  the  intestinal 
tract  exclusively.  From  decomposing  urine  a  larger  quantity  can 
be  obtained  than  from  fresh  urine.  This  is  no  doubt  owing  to  the 
fact  that  every  urine  contains  a  small  amount  of  carbohydrates, 
which  yield  fatty  acids  on  bacterial  decomposition.  Old  diabetic 
urine  is  hence  especially  rich  in  such  acids.  The  higher  fatty  acids 
are  normally  not  observed  in  the  urine,  but  traces  may  appear  under 
certain  pathological  conditions. 

Isolation  and  Quantitative  Estimation. — To  isolate  the  volatile 
fatty  acids  the  collected  urine  of  twenty-four  hours  is  acidified  with 
phosphoric  acid  in  the  proportion  of  10  :  100,  and  distilled  in  a  cur- 
rent of  steam  so  long  as  the  distillate  shows  an  acid  reaction.  This 
is  then  neutralized,  evaporated  to  dryness,  and  the  residue  extracted 
with  alcohol.  Traces  of  sodium  chloride,  which  are  formed  on  the 
addition  of  the  alkali,  owing  to  the  presence  of  a  little  hydrochloric 
acid  that  has  passed  over,  remain  behind.  The  alcoholic  solution 
is  then  evaporated  to  dryness,  the  residue  is  dissolved  in  a  little 
water,  acidified  with  sulphuric  acid,  and  set  aside  in  the  cold,  when 
traces  of  benzoic  acid  are  precipitated.  The  filtrate  is  neutralized 
with  a  solution  of  sodium  carbonate  and  extracted  with  ether,  which 
removes  the  phenols.  The  solution  is  now  acidified  with  sulphuric 
acid  and  is  again  distilled  in  a  current  of  steam,  when  the  volatile 
fatty  acids  pass  over.  Their  presence  can  be  established  according 
to  the  common  methods  of  analysis.  To  estimate  the  amount,  the 
final  distillate  is  neutralized  with  barium  hydrate,  evaporated  to  dry- 
ness, and  the  residue  weighed.  This  weight  less  that  of  the  barium, 
which  is  in  combination,  and  which  can  be  determined  as  barium 
sulphate,  after  incineration  and  extraction  with  dilute  hydrochloric 
acid,  indicates  the  amount  of  the  fatty  acids  in  general. 


THE  AROMATIC  OXY- ACIDS.  265 

^-oxybutyric  Acid. — This  acid  is  never  found  in  the  urine  under 
normal  conditions,  and  is  principally  met  with  in  the  severer  forms 
of  diabetes,  when  it  is  associated  with  the  presence  of  diacetic 
acid  and  acetone.  It  is  supposedly  derived  from  the  albumins  of 
the  tissues,  and  may  accordingly  also  appear  in  the  urine  in  various 
cachectic  conditions,  in  the  continued  fevers,  and  during  starvation. 
As  a  general  rule  it  is  found  in  combination  with  the  common  alkalies 
of  the  blood,  but  when  it  is  produced  in  especially  large  amounts  a 
corresponding  quantity  of  ammonia  is  furnished  by  the  body  to  effect 
its  neutralization.  It  may  happen,  however,  that  the  acid  formation 
exceeds  that  of  ammonia,  and  in  such  cases  the  free  acid  occurs  in 
the  urine,  and  can  also  be  demonstrated  in  the  blood  as  such. 
Symptoms  of  acid  intoxication  then  exist,  and  it  is  noteworthy  that 
in  such  cases  the  amount  of  carbonic  acid  in  the  blood  has  been 
found  markedly  diminished,  showing  that  the  alkaline  salts  are  not 
present  in  sufficient  amount  to  remove  the  carbonic  acid  from  the 
tissues. 

Of  the  immediate  antecedents  of  the  acid  nothing  is  known,  and 
if  formed  at  all  under  normal  conditions  it  is  manifestly  oxidized  at 
once  beyond  the  stage  of  diacetic  acid,  as  this  substance  also  is  only 
found  under  pathological  conditions.  Traces  of  acetone,  however, 
which  is  likewise  derived  from  oxybutyric  acid,  are  found  in  every 
urine. 

The  amount  of  oxybutyric  acid  which  may  occur  in  the  urine  is 
extremely  variable.  In  the  milder  cases  of  diabetes  it  is  usually 
absent ;  in  the  severer  forms,  however,  large  quantities  may  be 
found,  and  Kiilz  reports  that  in  three  cases  a  daily  elimination  of 
67,  100,  and  226  grammes,  respectively,  was  observed. 

The  chemical  relation  which  exists  between  /?-oxybutyric  acid, 
diacetic  acid,  and  acetone  is  seen  from  the  equations  : 

(1)  CH3.CH(OII).CH2.COOH  +  O  =  (CII3  CO)  ClI,,COOH  +  H20. 

0-oxy butyric  acid.  Diacelic  acid. 

(2)  (CIL.Co  MU'OOII  =CO(CIT3)2  +co2. 

Diacetic  acid.  Acetone. 

We  can  thus  readily  understand  that  in  certain  conditions  acetone 
may  be  found  in  the  urine  alone,  while  in  others  diacetic  acid,  and 
in  -till  others  ^-oxybutyric  acid  may  be  present  as  well. 

On  boiling  ,9-ox ybntyrie,  acid  in  aqueous  solution  with  dilute 
mineral  acids,  a-crotonic  acid  results,  and  it  is  thus  apparent  that 
this  acid  is  also  found  in  the  distillate,  when  urine  containing  the 
lir-t  i-  distilled  with  sulphuric  acid.  Otherwise,  however,  it  does 
CCUr.  The  reaction  which  takes  place  may  be  represented  by 
the  equation  : 

Ml    MI   nil    (  ll.COOir        MI,.MI  CHOOOH    |    11./) 

ixybutyric  acid.  o-crotontic  acid. 

Test. — A-  the  presence  of  oxybutyric  arid  presupposes  that  of 
diacetic  acid,  and  as  the  presence  of  the  latter  can  much  more 
readily  be  demonstrated  than  that  of  oxvbutvric  acid,  a  tesl  in  this 


266  THE   URINE. 

direction  should  always  precede  a  more  detailed  examination  (see 
below).  If  a  positive  reaction  is  thus  obtained,  any  sugar  that  may 
be  present  is  removed  by  fermentation.  The  liquid  is  cleared  by 
adding  neutral  acetate  of  lead,  when  the  filtrate  is  examined  with 
the  polarimeter.  Should  lsevorotation  now  be  observed,  the  presence 
of  oxybutyric  acid  is  rendered  very  probable.  To  demonstrate  this 
beyond  a  doubt,  the  liquid  is  evaporated  to  a  syrup,  treated  with  an 
equal  volume  of  concentrated  sulphuric  acid  and  distilled,  without 
cooling.  In  this  manner  the  oxybutyric  acid  is  decomposed  with 
the  formation  of  a-crotonic  acid,  which  is  accordingly  found  in  the 
distillate.  If  this  is  present  in  larger  amounts,  it  crystallizes  out  in 
the  distillate,  when  this  is  strongly  cooled,  and  may  be  identified  by 
its  melting-point,  72°  C.  Should  smaller  amounts,  however,  be 
present,  crystallization  does  not  occur.  In  this  case  the  distillate  is 
extracted  with  ether  by  shaking.  Traces  of  benzoic  acid  and  the 
phenols  are  thus  likewise  extracted,  but  if  then  the  residue  of  the 
ethereal  solution  is  washed  with  water  other  impurities  are  removed, 
and  the  crotonic  acid  remains. 

Diacetic  Acid. — From  what  has  been  said  above,  it  is  clear  that 
every  urine  which  contains  /3-oxybutyric  acid  must  also  contain 
diacetic  acid,  and  in  extreme  cases  both  may  be  found  in  the  blood 
as  such.  On  the  other  hand,  it  will  also  be  understood  that  diacetic 
acid  may  occur  in  the  urine  in  the  absence  of  ^-oxybutyric  acid, 
and  this  is  indeed  more  common. 

Tests. — As  diacetic  acid  is  rapidly  decomposed  on  standing,  it  is 
necessary  that  the  urine  should  be  as  fresh  as  possible,  when  it  is  to 
be  examined  in  this  direction.  To  this  end,  several  direct  tests  are 
available. 

Arnold's  Test. — This  test  is  the  most  reliable,  as  it  does  not 
respond  to  acetone  and  /9-oxybutyric  acid,  nor  to  the  common  anti- 
pyretics, salicylic  acid,  or  bile-pigment.  Highly  colored  urines, 
however,  should  first  be  filtered  through  animal  charcoal. 

Two  solutions  are  employed,  viz.,  a  1  per  cent,  solution  of  sodium 
nitrite,  and  a  solution  of  para-amido-acetophenon.  The  latter  is 
prepared  as  follows :  1  gramme  of  the  acetophenon  is  dissolved  in 
from  80  to  100  c.c.  of  distilled  water,  and  treated  drop  by  drop 
with  hydrochloric  acid  until  the  solution,  which  at  first  is  yellow, 
becomes  entirely  colorless  ;  an  excess,  however,  should  be  carefully 
avoided.  Before  using,  the  two  solutions  are  mixed  in  the  propor- 
tion of  one  part  of  the  nitrite  to  two  of  the  acetophenon  solution. 
A,  few  cubic  centimeters  of  the  urine  are  then  treated  with  an  equal 
volume  of  the  reagent,  and  a  few  drops  of  ammonia.  All  urines 
thus  give  a  more  or  less  well-marked  brownish-red  color  on  shaking, 
and  in  the  presence  of  much  diacetic  acid  an  amorphous  precipitate 
of  the  same  color  is  formed.  If  now  a  small  amount  of  the  colored 
solution  is  treated  with  an  excess  of  concentrated  hydrochloric  acid 
(10-12  c.c.  for  every  1  c.c),  a  beautiful  purplish-violet  color  de- 
velops if  diacetic  acid  is  present. 


THE  AROMATIC  OXY- ACIDS.  267 

Gerhakdt's  Test. — In  its  original  form  this  test  also  reacts 
with  the  common  antipyretics,  and  it  is  hence  necessary  to  isolate 
the  diacetic  acid  to  a  certain  degree.  To  this  end,  a  few  cubic  centim- 
eters of  the  urine  are  strongly  acidified  with  sulphuric  acid  and 
extracted  with  ether,  which  takes  np  the  acid.  The  extract  is  then 
shaken  with  a  few  cubic  centimeters  of  a  dilute  solution  of  the 
chloride  of  iron,  when  in  the  presence  of  diacetic  acid  the  aqueous 
layer  assumes  a  violet  or  Bordeaux-red  color.  This,  however,  is  not 
permanent,  and  soon  fades  on  boiling  the  solution. 

Acetone. — Traces  of  acetone,  varying  between  0.008  and  0.027 
gramme  in  the  twenty-four  hours  are  normally  found  in  the  urine. 
Larger  quantities  are  met  with  if  the  carbohydrates  are  withdrawn 
from  the  diet,  and  in  such  cases  from  0.2  to  0.7  gramme  may  be 
excreted  after  the  sixth  day.  If  then  carbohydrates  are  again 
ingested,  the  elimination  of  the  acetone  rapidly  reaches  its  origi- 
nal figure ;  the  ingestion  of  fats,  on  the  other  hand,  is  without 
effect  in  this  respect.  Acetonuria,  however,  is  essentially  a  patho- 
logical phenomonen,  and  is  observed  in  its  most  pronounced  form 
in  severe  cases  of  diabetes,  in  which,  as  I  have  stated,  it  is  fre- 
quently met  with  in  association  with  /3-oxybutyric  acid  and  diacetic 
acid.  Like  diacetic  acid,  however,  it  may  occur  in  the  absence  of 
oxybutyria  acid,  and  in  the  milder  forms  of  diabetes,  as  also  under 
normal  conditions,  it  may  be  present  alone.  But  while  its  import  is 
generally  the  same  as  that  of  its  immediate  antecedents,  it  appears 
that  it  may  originate  also  in  the  gastro-intestinal  tract  as  the  result 
of  some  abnormal  form  of  albuminous  putrefaction,  and  it  is  possi- 
ble, indeed,  that  the  so-called  asthma-acetonicnm  may  be  of  this 
origin.  That  small  amounts  are  formed  also  during  the  hydro- 
lytic  decomposition  of  the  albumins  with  boiling  mineral  acids  or 
the  caustic  alkalies,  has  been  mentioned,  v.  Jaksch,  further,  has 
shown  that  acetone  may  be  formed  as  a  by-product  during  the 
process  of  lactic  acid  fermentation,  and  it  has  been  suggested 
that  the  small  amounts  which  are  normally  met  with  in  the  urine 
may  originate  in  this  manner.  At  present,  however,  we  are  not 
in  a  position  to  speak  authoritatively  of  the  origin  of  these  normal 
trace-,  and,  pathologically  at  least,  we  can  thus  far  acknowledge 
but  our-  source  of  the  acetone,  viz.,  the  albumins  of  the  body 
tissues,  ami   secondarily    perhaps  the  circulating  albumins. 

Tests. — Should  diacetic  acid  be  demonstrated  in  the  urine,  the 
simultaneous  presence  of  acetone  may  be  directly  inferred.  If  this 
i-  not  the  case,  it  is  best  to  distill  from  250  <<•  500  c.c.  of  the  urine, 
after  the  addition  of  a  small  amount  of  phosphoric  acid,  and  to 
apply  the  following  tests  to  the  firsi  15  or  30  c.c.  (,f  the  distillate 
thai  has  passed  over. 

Legal's  Test. — A  few  cubic  centimeters  of  the  distillate  are 
treated  with  several  drops  of  a  freshly  prepared,  concentrated  solu- 
tion of  -odium  nitroprusside,  and  a  small  amount  of  a  dilute  solu- 


268  THE    URINE. 

tion  of  sodium  hydrate.  In  the  presence  of  acetone  a  red  color 
develops,  which  rapidly  fades  however,  but  is  replaced  by  a  beau- 
tiful carmin  or  purple  red  if  the  solution  is  treated  with  acetic 
acid  in  excess ;  on  standing,  this  turns  to  a  bluish  violet. 

Lieben's  Test. — On  adding  a  few  drops  of  a  dilute  solution  of 
iodopotassic  iodide  to  a  small  amount  of  the  distillate  that  has  been 
rendered  alkaline  with  sodium  hydrate  solution,  a  precipitate  of 
iodoform  develops  in  the  presence  of  acetone,  which  may  readily  be 
recognized  by  its  odor  on  warming  the  mixture.  This  test,  how- 
ever, is  not  conclusive,  as  other  substances,  such  as  alcohol,  give  the 
same  reaction. 

Gunning's  Test,  as  modified  by  Salkowski. — A  small 
amount  of  the  distillate  is  treated  drop  by  drop  with  freshly  pre- 
cipitated and  well-washed  mercuric  oxide  in  alcoholic  suspension 
until  a  portion  of  the  oxide  remains  undissolved.  The  liquid  is 
then  filtered,  and  the  filtrate  superposed  with  a  solution  of  ammo- 
nium sulphide,  when  in  the  presence  of  acetone  the  zone  of  contact 
assumes  a  grayish-black  color,  owing  to  the  formation  of  mercuric 
sulphide. 

Quantitative  Estimation. — The  quantitative  estimation  of  acetone 
is  best  conducted  according  to  the  method  of  Messinger,  as  modified 
by  Huppert.  It  is  based  upon  the  principle  underlying  Lieben's 
test,  viz.,  the  formation  of  iodoform  when  a  dilute  solution  of  iodo- 
potassic iodide  is  added  to  an  alkaline  solution  of  acetone.  By 
determining  the  amount  of  iodine  which  is  consumed  in  this  reac- 
tion the  corresponding  amount  of  acetone  can  then  be  calculated. 

One  hundred  c.c.  of  urine,  or  less  if  much  acetone  is  present,  as 
determined  by  Legal's  test  applied  directly  to  the  urine,  are  treated 
with  2  c.c.  of  a  50  per  cent,  solution  of  acetic  acid  and  distilled 
until  all  the  acetone  has  passed  over.  The  distillate  is  received  in 
a  bulb-tube  containing  water.  The  solution  which  thus  results  is 
treated  with  1  c.c.  of  a  12  per  cent,  solution  of  sulphuric  acid  and 
redistilled.  The  second  distillate  is  free  from  phenols.  To  it  a 
carefully  measured  quantity  of  a  one-tenth  normal  solution  of  iodine 
is  added  (10  c.c.  for  every  100  c.c.  of  urine),  together  with  a  50 
per  cent,  solution  of  sodium  hydrate,  until  the  iodoform  separates 
out.  After  shaking,  the  mixture  is  set  aside  for  a  few  minutes,  and 
then  acidified  with  concentrated  hydrochloric  acid.  If  iodine  is 
present  in  excess,  a  brown  color  thus  develops.  This  excess  is  then 
titrated  with  a  decinormal  solution  of  sodium  thiosulphate,  using 
starch  solution  as  a  final  indicator.  The  number  of  cubic  centim- 
eters emyloyed  in  this  titration  is  deducted  from  the  amount  of 
the  iodine  solution  added.  The  difference  multiplied  by  0.967  then 
indicates'  the  amount  of  acetone  in  the  100  c.c.  of  urine,  in  milli- 
grammes. 

Ehrlich's  Reaction. — When  normal  urine  is  treated  with  an  equal 
volume  of  a  saturated  solution  of  sulphanilic  acid  in   5   per  cent. 


THE  AROMATIC  OXY-ACIDS.  269 

hydrochloric  acid  to  which  a  0.5  per  cent,  solution  of  sodium 
nitrite  has  been  added  in  the  proportion  of  1  :  40,  and  the  mixture 
is  then  rendered  strongly  alkaline  with  ammonia,  a  more  or  less  well- 
marked  orange  color  develops.  Under  pathological  conditions,  on  the 
other  hand,  and  notably  in  typhoid  fever,  a  red  color  results  which 
varies  in  intensity  from  a  light  carmin  to  a  deep  garnet-red.  This 
reaction  is  known  as  Ehrlich's  diazo-reaction,  as  it  is  dependent 
upon  the  presence  of  a  diazo-compound  of  the  nature  of  diazo- 
benzene-sulphonic  acid  in  the  reagent.  This  results  through  an 
interaction  between  the  sodium  nitrite  and  the  sulphanilic  acid,  as 
represented  in  the  equations  : 

(1)    NaN02  +HC1     =HN02  +  NaCl. 


XNH2  .  N 

(2)    C6H4<  +HNOa  =  C6H4(         >N  +  2H20 


/ 


Sulphanilic  Diazo-benzene- 

acid.  sulphonic  acid. 

I  briefly  refer  to  the  reaction  at  this  place,  as  v.  Jaksch  has 
expressed  the  opinion  that  in  "  most "  cases  it  is  referable  to  the 
presence  of  acetone,  and  in  reality  represents  only  an  uncertain  test 
for  this  substance.  This  statement  I  wish  to  contradict  most 
emphatically,  as  I  have  been  able  to  show  beyond  a  doubt  that 
Ehrlich's  reaction  quite  commonly  occurs  at  a  time  when  abnormally 
large  amounts  of  acetone  cannot  be  demonstrated  in  the  urine,  and 
is  absent  in  many  diseases  in  which  a  marked  degree  of  acetonuria 
exists.  Normal  urines,  moreover,  in  which  traces  of  acetone  are 
(••instantly  found  do  not  give  the  red  color.  At  the  present  time  we 
are  in  total  ignorance  of  the  nature  of  the  substance  that  is  so  com- 
monly present  in  typhoid  urines,  and  which  reacts  with  diazo- 
benzene- sulphonic  acid  in  the  manner  indicated. 

Paralactic  Acid. — As  I  have  shown,  there  is  reason  to  believe 
that  the  greater  portion  of  the  nitrogen  which  is  set  free  in  the 
katabolism  of  the  various  tissues  appears  in  the  form  of  the  am- 
monium salt  of  paralactic  acid  and  is  transformed  into  urea  in 
the  liver.  Normally,  indeed,  paralactic  acid  is  not  found  in  the 
urine.  It  occurs,  however,  whenever  the  further  transformation  of 
the  ammonium  salt  is  impeded,  and  is  hence  met  with  in  various 
diseases  of  the  liver  which  arc  associated  with  an  extensive  destruc- 
tion of  the  hepatic  parenchyma,  as  also  in  conditions  in  which  the 
oxidation-processes  of  the  body  arc  impaired  in  general.  It  is  thus 
notably  met  with  in  acute  yellow  atrophy,  in  poisoning  with  phos- 
phorus and  carbon  monoxide,  in  long-continued  anaemic  conditions, 
see  also  page  223).  Smaller  amounts  have;  been  found  in 
soldier  after  forced  marches,  and  in  epileptic  patients  after  severe 
convulsive  seizures. 

Isolation. — To  isolate  the  substance  from  the  urine  the  following 
method  may  he  employed  as  suggested  by  Araki : 

The    collected    urine     of    twenty-four     hours     is     evaporated   to 


210  THE    UH1SE. 

about  50  or  60  c.c,  treated  with  ten  times  as  much  of  95  per  cent, 
alcohol,  and  set  aside  for  twelve  hours.  It  is  then  filtered  and  freed 
from  the  alcohol  by  distillation.  The  residual  fluid  is  acidified  with 
phosphoric  acid,  and  repeatedly  extracted  with  five  times  its  volume 
of  ether.  The  ethereal  extract  is  evaporated  and  the  remaining 
yellow  syrup  dissolved  in  a  little  water.  Any  hippuric  acid  which 
may  be  present  thus  separates  out  and  is  filtered  off.  The  filtrate  is 
now  treated  with  pure  lead  carbonate  in  substance,  heated  on  a 
water-bath  for  thirty  minutes,  and  filtered  on  cooling.  From  the 
filtrate  the  lead  is  removed  Joy  means  of  hydrogen  sulphide,  and  the 
excess  of  the  latter  by  gently  warming  on  a  water-bath.  The 
fluid  is  then  concentrated  to  a  thick  syrup  and  extracted  with  ether. 
The  ethereal  solution  is  evaporated  and  the  residue  boiled  for  some 
time  with  water  and  an  excess  of  zinc  carbonate.  The  mixture 
is  filtered  while  hot,  concentrated  to  a  small  volume,  and  then  set 
aside  in  the  cold  after  adding  a  little  alcohol.  The  zinc  salts  of 
both  paralactic  acid  and  the  common  optically  inactive  lactic  acid 
which  may  also  be  present  in  traces,  then  crystallize  out.  They 
can  be  separated  from  each  other  by  treating  with  absolute  alcohol, 
in  which  the  latter  is  insoluble.  It  must  be  noted  that  the  solu- 
bility of  the  paralactate  is  also  slight  (1  :  1100),  so  that  it  is 
necessary  to  add  a  large  amount  of  alcohol.  In  order  to  prevent 
confusion  with  the  aromatic  oxy-acids  of  the  urine,  the  lactate 
crystals  should  now  be  further  identified,  which  is  most  conveniently 
done  by  estimating  the  water  of  crystallization.  The  paralactate 
contains  two  molecules  of  this,  which  escapes  at  105°  C,  and  at  this 
temperature  the  weight  of  the  crystals  should  therefore  diminish  12.9 
per  cent.  The  salt,  moreover,  like  its  acid,  is  laevorotatory,  while 
the  common  lactate  is  optically  inactive. 

Leucin  and  Tyrosin. — Leucin  and  tyrosin,  according  to  some 
observers,  are  normally  present  in  the  urine  in  traces.  By  others  this 
is  denied,  and  I  must  admit  that  I  have  never  found  either  of  the 
substances  under  normal  conditions.  In  certain  diseases  of  the 
liver,  however,  in  which  extensive  destruction  of  the  hepatic  cells 
is  going  on,  both  may  be  found.  But  it  is  noteworthy  that 
while  in  acute  yellow  atrophy  this  is  a  common  occurrence  after 
the  first  week  of  the  disease,  in  acute  phosphorus  poisoning  they 
are  usually  not  found.  Thus  far  we  have  no  adequate  explana- 
tion to  offer  for  this  difference,  and  we  are  in  ignorance,  more- 
over, of  the  origin  of  the  bodies  in  question  in  the  former 
disease.  "We  have  seen  that  as  a  general  rule  at  least  the  greater 
portion  of  the  tissue  nitrogen  which  is  set  free  during  the  process 
of  metabolism  is  carried  to  the  liver  in  the  form  of  ammonium 
paralactate,  and  there  is  no  evidence  to  show  that  this  may  be 
transformed  into  either  leucin  or  tyrosin.  We  see,  in  fact,  that  in 
extensive  hepatic  disease  ammonium  lactate  appears  in  the  urine. 
Whether  or  not  in  acute  yellow  atrophy  leucin  and  tyrosin  are  also 
set  free  in  the  tissues  in  general,  we  do  not  know.     In  the  liver,  it  is 


THE  NEUTRAL  SULPHUR   OF  THE   URINE.  271 

true,  both  are  then  met  with  in  large  amount,  but  we  may  readily 
suppose  that  the  substances  may  have  originated  here  directly,  and 
possibly  as  a  result  of  some  fermentative  action.  This,  indeed, 
appears  the  most  likely  explanation. 

Isolation. — If  leiicin  and  tyrosin  are  present  in  the  urine  in  small 
amounts,  they  are  held  in  solution.  In  the  presence  of  larger 
amounts  the  tyrosin  may  separate  out,  and  can  then  be  isolated  from 
the  sediment  and  identified  as  described.  Leucin,  however,  is  rarely 
found  in  this  manner,  and  remains  in  solution  even  though  very 
large  quantities  are  eliminated. 

To  demonstrate  both  when  they  are  held  in  solution,  it  is  some- 
times only  necessary  to  concentrate  a  small  amount  of  urine  on  a 
water-bath  and  to  examine  the  residual  syrup  with  the  microscope. 
Otherwise  it  is  advisable  to  precipitate  the  collected  urine  of 
twenty-four  hours,  after  removing  any  albumin  that  may  be  present, 
with  basic  lead  acetate.  The  filtrate  is  then  freed  from  lead  with 
hydrogen  sulphide,  evaporated  to  a  thick  syrup,  and  set  aside  for 
crystallization.  Tyrosin  and  leucin  can  then  be  demonstrated  by  a 
microscopical  examination  and  identified  in  the  usual  manner  (see 
page  188). 

THE  NEUTRAL  SULPHUR  BODIES  OF  THE  URINE. 

In  the  section  on  the  mineral  constituents  of  the  urine  I  pointed 
out  that  the  greater  portion  of  the  sulphur  which  is  set  free  during 
the  metabolism  of  the  nitrogenous  constituents  of  the  body  is  elimi- 
nated in  the  urine  in  a  completely  oxidized  form.  A  much  smaller 
fraction,  however,  escapes  oxidation,  and  appears  in  the  urine  as 
so-called  neutral  sulphur.  Normally  this  constitutes  about  12-15 
per  cent,  of  the  total  amount.  Of  the  individual  components  which 
go  to  make  up  this  neutral  sulphur  comparatively  little  is  known, 
ami  it  appears,  moreover,  that  their  character  may  be  different  in 
different  animals.  In  the  urine  of  eats,  and  less  constantly  in  that 
of  dogs,  traces  of  thiosulphatea  are  thus  found,  while  in  man  this  is 
normally  absent,  ami  in  disease  even  thiosulphuric  acid  has  been  found 
in  only  one  instance— in  typhoid  fever.  Ethyl  sulphide,  or  a  body 
which  gives  rise  to  its  formation  when  the  urine  is  treated  with 
lime-water,  is  thus  similarly  not  found  in  the  urine  of  man,  while  it 
is  apparently  a  constant  constituent  of  that  of  dogs. 

Ol  the  normal  constituents  of  the  neutral  sulphur  which  is  found 
in  human  urine,  only  two  are  actually  known.  These  are  the  sulpho- 
cyanides,  which  are  found    in    small  quantities   in  tin-   saliva  and  the 

trie   juice,  and  cystein,  or   a    body  which    is   closely  related    to   it. 

Whether  or  not  taurocarbaminie  acid  is  also  constantly  present  has 
not  as*  yet  been  determined.     I   have   shown,   however,  that   to  a 

certain   extent  at   least     tanrin    is  eliminated    in  this  form  when  given 

by  the  month.  In  cases  of  obstructive  jaundice,  moreover,  or  after 
ligation  of  the  common  dud  in  dogs,  the  neutral  sulphur  may  increase 


272  THE   URINE. 

to  40  per  cent,  of  the  total  amount,  and  it  is  known  that  in  such 
cases  taurocarbaminic  acid  is  constantly  present.  Its  formation  may 
be  represented  by  the  equation  : 

/C2H4.NH2  yNH2  /NH2 

S02<  +CO<  =CO< 

xOH  \NH,  \NH.C2H4.S02.O.NH4. 

Taurin.  Urea.  Ammonium  taurocarbamate. 

In  all  probability  this  synthesis  is  effected  in  the  kidneys.  In 
rabbits,  on  the  other  hand,  taurin  is  largely  oxidized  to  sulphuric 
acid,  while  a  small  portion  appears  as  thiosulphuric  acid.  Of  the 
origin  of  taurin,  as  I  have  stated,  nothing  definite  is  known. 

Cystein,  on  the  other  hand,  is  apparently  derived  from  the 
loosely  combined  sulphur  of  the  albumins,  and  probably  represents 
an  intermediary  product  of  oxidation,  which  is  normally  further 
oxidized  to  sulphuric  acid.  Traces,  however,  apparently  escape  this 
destruction  and  normally  appear  in  the  urine.  As  a  matter  of  fact, 
cystein  is  not  readily  oxidized  within  the  body,  and  in  dogs  one- 
third  of  the  ingested  amount  reappears  as  such.  Following  the 
administration  of  chlorine,  bromine,  or  iodine  substitution-products 
of  the  benzols,  moreover,  a  diminished  elimination  of  sulphuric  acid 
occurs,  and  in  place  of  this  we  meet  with  a  conjugate  glucuronate, 
which  contains  the  greater  portion  of  the  lacking  sulphur.  The 
product  which  thus  results  can  readily  be  decomposed,  with  the 
formation  of  glucuronic  acid  and  chlorophenyl-mercapturic  acid, 
which  latter  manifestly  contains  the  cystein  group,  as  is  seen  from 
the  formulae : 


CH3 

CH3 

NH2X  | 

CH3.CO.NHv    | 
C6H4C1.S^^  | 

COOH 

COOH 

On  decomposition  it  yields  acetic  acid  and  chlorophenyl-cystein, 
as  shown  in  the  equation  : 


CHS 

CH3 

CH3.CO.NH     | 

NH2v     | 
C6H4C1.S/    | 

>C        +  H20  = 
C6H4C1.S/  | 

=  CH3.COOH  + 

COOH 

COOH 

The  amount  of  cystein  which  is  normally  present  in  the  urine 
probably  does  not  exceed  0.015  gramme  in  the  twenty-four  hours. 
Larger  quantities  are  found  in  phosphorus  poisoning,  which  further 
suggests  the  occurrence  of  the  substance  as  an  intermediary  product 
in  the  normal  metabolism  of  the  organic  sulphur,  as  we  know  that 
in  such  cases  the  oxidation-processes  of  the  body,  as  a  whole,  are 
much  impaired. 


THE  NEUTRAL  SULPHUR   OF  THE   URISE.  273 

On  exposure  to   the  air  cystei'n  is  transformed  into  cystin,  as  is 
shown  in   the  equation  : 


Cystin. 


This  transformation  is  of  special  interest,  owing  to  the  fact  that 
under  certain  conditions  cystin  also  may  appear  in  the  urine,  while 
normally  it  is  absent.  It  is  noteworthy,  moreover,  that  cystinuria 
can  scarcely  be  regarded  as  a  pathological  phenomenon,  although  it 
mav  occur  in  association  with  a  definite  disease.  More  commonly 
it  is  observed  in  otherwise  normal  individuals,  and,  like  alkaptonuria, 
it  may  persist  for  a  lifetime,  and  may  occur  in  families.  It  may 
hence  be  regarded  as  a  metabolic  anomaly  in  which  the  oxidation 
of  the  loosely  combined  sulphur  is  diminished,  or  perhaps  even 
suspended.  Very  curiously,  it  has  further  been  observed  that 
cystin uria  is  quite  constantly  accompanied  by  the  appearance  of 
cadaverin  in  the  urine,  while  in  two  instances  at  least  putrescin 
also  was  found.  Some  observers  have  hence  suggested  that  a 
genetic  relationship  may  exist  between  the  two  conditions,  and 
that  the  formation  of  the  diamins  may  be  the  primary  factor.  But 
while  most  observers  assume  that  the  diaminuria  is  referable  to 
the  presence  of  certain  specific  organisms  in  the  intestinal  canal, 
which  are  usually  absent,  I  am  personally  inclined  to  regard  the 
formation  of  the  diamins  also  as  a  metabolic  anomaly,  and  suppose 
that  both  conditions  are  the  outcome  of  a  third  factor,  as  yet  un- 
known, but  which  no  doubt  operates  within  the  tissues  directly. 
The  possible  formation  of  diamins  in  the  absence  of  micro-organ- 
isms can  indeed  no  longer  be  doubted  (see  page  73). 

The  amount  of  cystin  which  may  be  met  with  in  the  urine  is 
extremely  variable.  On  some  days  traces  only  are  found,  while  on 
others  the  elimination  may  exceed  1  gramme  in  the  twenty-four 
hours.  That  the  total  amount  of  the  neutral  sulphur  is  then  also 
proportionately  increased  is  of  course,  self-evident. 

Outside  of  the  urine  cystin  has  been  encountered  in  onlv  a  few 
instances.  Cloetta  thus  ''hums  to  have  obtained  the  substance 
from  the  kidneys  of  the  ox.  Seherer  found  it  once  in  the  liver 
of  a  typhoid-fever  patient,  and  Drechsel  isolated  the  body  from 
th'-  liver  of  a  horse  and  a  porpoise.  Kiilz  further  claims  to  have 
found  cystin  among  the  decomposition-products  of  fibrin  on  one 
ision  where  the  digestion  was  effected  with  pancreas.  Together 
with   Dr.  Amberg,  however,  1  have  been  unable  to  confirm   Kiilz's 

temenl  in  a  series  of  experiments  undertaken  in  my  laboratory. 
Of  late,  Morner   has    shown   that   cystin    results  on   decomposing 

the    keratine  of    horn    shavings  with    mineral    acid-. 

[Jnlese  the  cystin  i-  found  directly  in  a  urinary  sediment,  its  pres- 

18 


274  THE    UEINE. 

ence  will  scarcely  be  suspected.  If,  however,  a  urine  develops  a 
marked  odor  of  hydrogen  sulphide  on  standing,  it  is  well  to  add  an 
excess  of  acetic  acid  and  to  examine  the  sediment  somewhat  later. 
The  characteristic  hexagonal  platelets  of  cystin  may  then  at  times 
be  found,  and  cau  be  recognized  from  their  solubility  in  ammonia 
and  hydrochloric  acid,  while  they  are  insoluble  in  acetic  acid, 
water,  alcohol,  and  ether.  But  sometimes  this  procedure  does  not 
lead  to  the  desired  end,  even  though  a  decided  increase  in  the 
amount  of  neutral  sulphur  is  observed,  and  hydrogen  sulphide  is 
formed  in  abundance  on  standing.  Whether  or  not  it  may  then 
be  justifiable  to  refer  this  increase  of  the  neutral  sulphur  to  the 
presence  of  cystin,  is  questionable. 

Clinically  cystin  is  of  interest  in  so  far  as  its  continued  appear- 
ance in  the  urine  may  be  regarded  as  a  probable  precursor  of  the 
formation  of  cystin  gravel  or  calculi ;  and  we  find,  as  a  matter  of 
fact,  that  this  occurs  in  a  very  considerable  proportion  of  all  cases. 

A  quantitative  estimation  of  the  cystin,  isolated  as  such,  is  not  as 
yet  possible.  When  it  is  found  in  a  sediment  the  crystals  may  be  col- 
lected and  weighed.  But  as  a  variable  amount  remains  in  solution, 
even  after  the  addition  of  much  acetic  acid  to  the  urine,  it  is  further 
necessary  to  estimate  the  total  amount  of  neutral  sulphur  that  re- 
mains, when  an  excess  beyond  the  average  figures  may  be  referred  to 
cystin,  and  the  result  added  to  that  obtained  directly. 

Isolation. — As  the  synthesis  of  cystin  has  not  as  yet  been  effected, 
we  are  generally  obliged  to  rely  upon  cystin  concretions  for  purposes 
of  study.  If  such  material  is  inaccessible,  we  may  prepare  the  sub- 
stance from  horn  shavings  by  decomposing  the  contained  keratins 
with  mineral  acids.  For  the  isolation  of  the  body  from  the  result- 
ing decomposition-products,  however,  I  must  refer  the  reader  to 
Morner's  article. 

Properties. — Several  varieties  of  cystin  apparently  exist,  of  which 
one  islsevorotatory,  another  dextrorotatory,  while  a  third  is  optically 
inactive.  The  common  cystin  which  is  found  in  the  urine  belongs 
to  the  lsevorotatory  type.  It  crystallizes  in  colorless,  hexagonal 
platelets,  which  are  quite  characteristic.  They  are  soluble  in  solu- 
tions of  the  alkaline  hydrates,  in  ammonia,  and  the  mineral  acids. 
In  water,  alcohol,  ether,  and  acetic  acid  the  substance  is  insoluble, 
as  also  in  solutions  of  ammonium  carbonate,  and  it  is  for  this 
reason  that  cystin  is  apt  to  crystallize  out  from  decomposing  urines 
if  it  was  previously  present  in  solution  only. 

Structurally,  cystin  is  the  disulphide  of  cystei'n,  which  in  turn  is 
«-amido-thioiactic  acid.  On  reduction  it  is  transformed  into  cyste'in, 
as  shown  in  the  equation  : 

CH3  CH3  CH3 

|    /NH2  NH2.     |  NH2X    I 

c/  \C  +2H=2  >C 

|   \S S/    |  SH  /  I 

COOH  COOH  COOH 

Cystin.  Cyste'in. 


THE   CARBOHYDRATES.  275 

On  heating  the  substance  on  platinum  foil  it  does  not  melt,  but 
ignites  and  burns  with  a  bluish-green  flame ;  at  the  same  time 
a  peculiar,  penetrating  odor  develops.  It  does  not  give  the  murexid 
reaction.  When  boiled  with  caustic  alkali  it  is  decomposed  and 
the  sulphur  liberated  as  a  sulphide.  With  benzoyl  chloride,  in  the 
presence  of  an  excess  of  caustic  alkali,  it  forms  benzoyl-cystin,  and 
is  thus  precipitated  as  a  sodium  salt  in  the  form  of  fine  lustrous 
platelets,  which  are  readily  soluble  in  water,  but  insoluble  in  solu- 
tions of  the  caustic  alkalies.  Upon  the  addition  of  an  acid  to  such 
a  solution,  benzoyl-cystin  separates  out  as  such.  It  is  soluble  in 
alcohol  and  alcohol-ether,  slightly  so  in  pure  ether,  and  almost  insolu- 
ble in  water.  Its  needle-like  crystals  melt  at  156°-158°  C.  The 
formation  of  benzoyl-cystin  may  be  expressed  by  the  equation  : 

CH3  CH3 

2C,-H5.C0C1  +  C(  >C  = 

Benzoyl  I    \S S'     I  •« 

chloride.  coofj  C00H 

Cystin. 

CH3  CH3 

I   /NH(C6H5.CO)    (C6H5.CO)NHN    | 

C<  >C  +  2HC1 

I      S B/  I 

CUOH  COOH 

Benzoyl-cystin. 

On  boiling  with  concentrated  hydrochloric  acid,  benzoyl-cystin  is 
decomposed  with  the  formation  of  benzoic  acid  and  cystin. 

Quantitative  Estimation  of  the  Neutral  Sulphur — In  one 
portion  of  the  urine  the  oxidized  sulphur,  viz.,  the  mineral  and  the 
conjugate  sulphates,  is  estimated  as  has  been  described.  In  a 
second  portion  the  total  sulphur  is  then  ascertained  as  follows:  100 
c.c.  of  urine  are  treated  with  12  grammes  of  a  mixture  of  sodium 
and  potassium  carbonate  (11  :  14),  and  evaporated  to  dryness  in  a 
platinum  dish.  The  residue  is  thoroughly  fused,  and  on  cooling 
extracted  with  hot  water.  The  carbonaceous  residue  is  filtered  off, 
washed  with  hot  water,  and  filtrate  and  washings  treated  with  a 
few  crystals  of  potassium  permanganate.  After  heating  for  fifteen 
minutes  (a  little  more  of  the  permanganate  must  be  added  if  the 
solution  becomes  decolorized),  and  concentrated  hydrochloric  acid  is 
added  until  the  liquid  is  distinctly  acid.  It  is  then  brought  to  the 
boiling-point,  treated  with  _!<>  c.c.  of  a  hot  saturated  solution  of 
barium  chloride,  when  the  barium  sulphate  which  is  thus  formed  Is 
estimated  as  usual  (see  page  220).  The  difference  between  the  two 
results  indicates  the  amount  of  the  neutral  sulphur. 

THE  CARBOHYDRATES. 

The  carbohydrates  which  may  be  found  in  the  urine  comprise 
glucose,  laevulose,  laiose,  maltose,  lactose,  dextrin,  animal  gum,  and 
certain  pentoses.     Of  these,  truce-  of  glucose,  dextrin,  animal  gum, 


276  THE    URINE. 

and  possibly  also  pentoses,  may  be  found  at  all  times.  Their 
amount,  however,  is  normally  so  small  that  their  presence  cannot  be 
recognized  by  the  common  tests.  Larger  amounts  of  carbohydrates 
are  found  in  health  only  during  the  puerperal  state,  and  in  the 
course  of  lactation,  when  lactose  is  commonly  present.  Otherwise 
the  elimination  of  sugar  in  amounts  which  can  be  demonstrated  by 
the  ordinary  tests  must  be  regarded  as  abnormal. 

Glucose. — As  I  have  indicated,  glucose  appears  in  the  urine 
whenever  its  amount  in  the  blood  exceeds  3  pro  mille.  This, 
however,  occurs  only  under  abnormal  conditions,  and  in  the  pres- 
ence of  small  amounts  the  kidneys  are  manifestly  capable  of 
preventing  its  passage  into  the  urine.  Under  certain  conditions, 
however,  this  power  is  apparently  lost,  and  we  find,  as  a  matter 
of  fact,  that  following  the  administration  of  phlorhizin  glucosuria 
occurs,  although  the  percentage  of  sugar  is  not  increased  in  the 
blood.  Whether  or  not  such  an  insufficiency  on  the  part  of  the 
kidneys  may  also  occur  spontaneously  we  do  not  know.  As  a  gen- 
eral rule,  however,  glucosuria  is  associated  with  a  hypergluchsemia. 
This  may  result  if  unduly  large  amounts  of  sugar  reach  the  liver, 
so  that  the  organ  is  incapable  of  transforming  the  entire  quantity 
into  glycogen,  and  I  have  pointed  out  that  the  functional  capacity 
of  the  liver  in  this  respect  is  of  a  much  lower  order  than  the 
ability  of  the  intestinal  epithelium  to  transform  polysaccharides 
and  disaccharides  into  glucose.  The  extent  to  which  the  liver 
can  normally  transform  glucose  into  glycogen  seems  to  vary 
with  different  individuals.  Generally,  glucosuria  occurs  when 
the  amount  of  sugar  exceeds  200  grammes.  There  are  many 
individuals,  however,  in  which  this  occurs  following  the  admin- 
istration of  only  150  grammes,  and  there  are  others  in  which  the 
ingestion  of  250  grammes  does  not  cause  glucose  to  appear  in 
the  urine.  Glucosuria  following  the  ingestion  of  100  grammes  of 
grape-sugar  is  now  regarded  as  abnormal,  and  there  is  reason  to 
believe  that  the  hepatic  insufficiency  thus  manifested  may  be  of  the 
type  of  a  mild  form  of  diabetes.  The  amount  of  sugar  which  then 
appears  in  the  urine  rarely  exceeds  3  per  cent.  The  glucosuria, 
moreover,  is  only  temporary,  and  disappears  as  soon  as  the  ingestion 
of  sugar  (viz.,  starches)  is  diminished.  Between  this  form  of  glu- 
cosuria and  the  common  form  of  diabetes,  in  which  practically  no 
sugar  can  be  utilized  by  the  body,  but  in  which  the  elimination 
ceases  as  soon  as  carbohydrates  are  withdrawn  from  the  diet,  all 
gradations  may  occur.  These  forms  are  now  generally  regarded  as 
referable  to  a  hepatic  insufficiency  of  whatever  origin.  Quite  differ- 
ent from  diabetes  of  this  character,  on  the  other  hand,  is  the  type 
in  which  the  glucosuria  continues  although  no  sugars  are  ingested. 
In  such  cases  a  hepatic  insufficiency  need  not  necessarily  exist, 
and  there  is  evidence  to  show  that  in  these  forms  the  formation 
of  glycogen  may  still  occur.  We  must  therefore  assume  that 
other  organs    are  primarily  involved,  and  there  is  every  reason  to 


THE  CARBOHYDRATES.  277 

suppose  that  the  metabolism  of  muscle-tissue  is  here  principally 
at  fault,  and  that  the  tissue  has  lost  the  power  of  decomposing 
the  sugar  which  reaches  it  from  the  liver.  As  a  consequence, 
increased  destruction  of  muscle-tissue  occurs,  as  the  inability  on 
the  part  of  these  structures  to  decompose  sugar  amounts  to  the 
same  as  though  no  sugar  were  present  at  all.  The  body  therefore 
liberates  the  carbohydrate  groups  of  its  albumins  to  supply  the 
apparent  deficit,  and  thus  further  increases  the  hypergluchsernia 
and  the  resulting  glucosuria.  We  accordingly  find  that  even 
though  the  carbohydrates  have  been  withdrawn  from  the  food 
sugar  still  appears  in  the  urine.  The  increased  destruction  of  the 
tissue  albumins  is  in  such  cases  sufficiently  apparent  from  the  pro- 
gressive loss  of  flesh  which  is  so  constantly  observed.  Of  the  causes 
which  are  operative  in  bringing  about  the  muscular  insufficiency  as 
regards  the  decomposition  of  sugar,  we  know  but  little.  That  cer- 
tain nervous  influences  may  here  be  at  work  is  probable,  and  we 
know,  as  a  matter  of  fact,  that  injury  to  a  certain  region  in  the  floor 
of  the  fourth  ventricle  is  invariably  followed  by  the  appearance  of 
glucose  in  the  urine.  But,  on  the  other  hand,  we  may  also  imagine 
that  the  normal  decomposition  of  the  sugar  is  prevented  owing  to  the 
absence  of  some  such  ferment  as  the  glucolytic  ferment  of  Lepine, 
and,  as  has  been  pointed  out,  this  ferment  is  in  all  likelihood  formed 
in  the  pancreas.  In  support  of  this  view  is  the  fact  that  after 
extirpation  of  the  pancreas  death  invariably  results  with  symptoms 
whicli  are  practically  identical  with  those  seen  in  the  gravest  types 
of  diabetes.  Ligation  of  the  duct  does  not  produce  this  effect ; 
and  it  is  noteworthy,  moreover,  that  the  glucosuria  disappears  when 
pieces  of  the  pancreas  are  transplanted  under  the  skin  or  when 
fresh  raw  pancreas  is  given  with  the  food.  Within  the  past  ten 
years  it  has  been  found  that  in  a  not  inconsiderable  number  of 
cases  of  diabetes  degenerative  lesions  can  be  demonstrated  in  the 
pancreas,  and  there  can  be  no  doubt  at  the  present  time  that  a 
certain  percentage  of  cases  are  directly  referable  to  such  origin. 
In  the  milder  forms,  on  the  other  hand,  an  insufficiency  on  the 
part  of  the  muscle-tissue  manifestly  does  not  exist,  as  it  is  possible 
to  prevent  the  occurrence  of  glucosuria,  temporarily  at  least,  if  the 
demand  for  sugar  is  increased  by  abundant  muscular  exercise. 
That  a  hepatogenic  diabetes  finally  may  coexist  with  a  myogenic 
form,  cannot   be-  doubted. 

This  i-.  however,  not  the  plaee  to  enter  into  a  detailed  account  of 
the  mechanism  by  which  glucosuria  is  produced,  and  for  further 
information,  and  lor  a  consideration  of  the  various  pathological  con- 
dition- under  which  BUgar  may  be  found  in  the  urine,  the  reader  is 
referred  to  other  works. 

The  amount  of*  sugar  which  may  be  present  in  the  urine  under 
pathological  condition-  i-  exceedingly  variable.  On  the  one  hand, 
traces  only  may  he  found,  which  may  be  normal  ;  while,  on  the  other 
hand,  the  daily  excretion   may  exceed    1000   grammes.     In  diabetes 


278  ,  THE   URINE. 

an  elimination  of  from  3  to  6  per  cent,  in  an  amount  of  urine 
varying  between  3000  and  6000  c.c.  may  be  regarded  as  moderate. 

Tests  for  Sugar. — Simple  tests  by  means  of  which  glucose  can  be 
demonstrated  directly  in  the  urine  as  such  are,  unfortunately,  not 
available.  Other  sugars,  it  is  true,  enter  into  consideration  only 
under  exceptional  conditions,  but  if  it  is  desired  to  prove  that  the 
substance  which  gives  the  common  sugar  reactions  is  actually  glu- 
cose, a  more  detailed  examination  is  necessary.  Some  of  these  tests, 
moreover,  may  be  simulated  by  substances  which  are  not  carbo- 
hydrates, and  deductions  as  to  the  presence  or  absence  of  sugar  are 
hence  only  warrantable  when  these  can  be  excluded.  If  albumins 
are  present,  they  must  first  be  removed. 

Nylander's  Test. — This  test  is  to  be  preferred  to  the  more 
common  one  of  Trommer,  as  the  reagent  does  not  react  with  uric 
acid,  kreatinin,  or  homogentisinic  acid.  With  many  of  the  conju- 
gate glucuronates,  however,  a  reduction  is  observed,  and  it  is  hence 
necessary  to  eliminate  this  source  of  error  when  a  positive  reac- 
tion is  obtained,  or  to  apply  additional  tests  in  which  this  possi- 
bility does  not  enter  into  consideration. 

The  reagent  is  prepared  as  follows  :  4  grammes  of  the  tartrate 
of  potassium  and  sodium,  together  with  2  grammes  of  subnitrate  of 
bismuth  and  10  grammes  of  sodium  hydrate  are  placed  in  90  c.c. 
of  water.  The  solution  is  heated  to  the  boiling-point,  filtered  on 
cooling,  and  is  then  ready  for  use.  It  is  kept  in  a  dark-colored 
bottle. 

A  few  cubic  centimeters  of  the  urine  are  treated  with  the  reagent, 
in  the  proportion  of  11  :  1,  and  boiled,  when  in  the  presence  of 
sugar  a  reduction  of  the  subnitrate  of  bismuth  to  bismuthous  oxide, 
or  even  to  the  metallic  form,  occurs.  As  a  consequence  the  mixture 
assumes  a  grayish,  dark-brown,  or  black  color,  and  on  standing  the 
precipitated  oxide  or  metal  settles  to  the  bottom  together  with  the 
earthy  phosphates. 

Fehling's  Test. — This  is  merely  a  modification  of  the  older 
Trommer's  test.  The  reagent  consists  of  two  solutions,  viz.,  one 
containing  34.64  grammes  of  copper  sulphate  in  500  c.c.  of  water, 
while  the  other  is  prepared  by  dissolving  173  grammes  of  tartrate 
of  potassium  and  sodium  and  125  grammes  of  caustic  soda  in  a  like 
amount  of  water.  Before  using,  equal  parts  of  the  two  solutions 
are  mixed  and  diluted  with  four  times  as  much  water.  A  few  cubic 
centimeters  of  the  resulting  reagent  are  boiled  and  treated  with  a 
small  amount  of  the  urine,  when  in  the  presence  of  sugar  yellow 
cuprous  hydroxide  or  red  cuprous  oxide  separates  out,  and  on  stand- 
ing settles  to  the  bottom.  After  the  addition  of  the  urine  the  solu- 
tions should  no  longer  be  boiled,  but  may  be  held  near  the  flame 
for  a  few  moments.  Unless  this  precaution  is  taken,  fallacious 
results  are  often  obtained,  as  uric  acid,  and  kreatinin  more  especially, 
may  cause  a  partial  reduction  of  the  copper  solution  on  prolonged 
boiling.     The  test  at  best  is  open  to  many  objections.     Conjugate 


THE  CARBOHYDRATES.  279 

glucuronates  and  homogentisinic  acid  likewise  give  a  positive  reac- 
tion, and  ammonia,  if  present  beyond  traces,  may  hold  in  solution 
any  cuprous  oxide  that  may  be  referable  to  very  small  amounts  of 
sugar. 

Fermentation  Test. — This  test,  when  controlled  by  Nylander's 
test,  is  the  most  satisfactory  one.  To  this  end,  a  little  compressed 
yeast  is  shaken  with  about  20  c.c.  of  urine,  and  the  mixture  is 
placed  in  a  saccharimetric  tube,  such  as  that  devised  by  Lohnstein 
or  Einhorn.  On  standing  at  the  ordinary  temperature  of  the  room, 
or,  still  better,  at  37°  C,  fermentation  occurs  if  glucose  is  present, 
and  the  liberated  carbon  dioxide  collects  at  the  top  of  the  tube. 
In  any  case,  however,  two  controls  should  be  made,  viz.,  one  to 
determine  that  the  yeast  is  active,  and  another  with  normal  urine. 
If  a  small  amount  of  sugar  is  present,  it  may  happen  that  the 
resulting  carbon  dioxide  is  absorbed.  If  in  such  a  case  Nylander's 
test  first  gave  a  positive  reaction,  but  no  longer  reacts  after  fermenta- 
tion is  complete  (in  twelve  to  twenty-four  hours),  the  presence  of 
sugar  may  be  inferred.  If,  on  the  other  hand,  no  fermentation 
occurs,  and  Xylander's  test  still  gives  a  positive  result,  we  may  con- 
clude that  the  reaction  is  due  to  the  presence  of  a  non-fermentable 
reducing  substance. 

Phenylhydrazin  Test. — As  has  been  pointed  out,  all  mono- 
saccharides and  some  of  the  disaccharides,  such  as  maltose,  isomal- 
fcose,  and  lactose,  form  compounds  with  phenylhydrazin  which  are 
known  as  osazons  (see  page  55).  The  resulting  bodies  are  all  very 
similar,  but  may  be  distinguished  from  each  other  by  the  melting- 
point  of  their  crystals,  and  to  some  extent  also  by  their  microscopical 
appearance.  With  free  glucuronic  acid  a  similar  compound  may 
be  obtained,  according  to  Thierfelder,  which  may  also  be  recognized 
by  its  melting-point,  while  the  conjugate  glucuronates  are  inactive  in 
this  respect.  Pentoses  likewise  give  rise  to  the  formation  of  osa- 
zons,  but  the  melting-point  of  the  resulting  crystals  serves  to  distin- 
guish these  also  from  the  osazons  of  the  hexoses.  As  a  general 
rule,  however,  neither  the  pentoses  nor  glucuronic  acid  interferes 
with  the  reliability  of  the  test.  If  doubt  should  arise,  a  special 
examination  should  be  made  to  ascertain  whether  pentoses  or  glu- 
curonates arc  present  in  amounts  sufficient  to  react  with  the  reagent. 
A  further  objection  to  the  phenylhydrazin  test  has  been  urged  on 
the  basis  that  its  delicacy  is  such  that  a  positive  reaction  is  obtained 
even    under  normal   condition-.      This,  however,  I  must  deny. 

The  tes<  is  conveniently  conducted  as  follows;  5  drops  of  pure 
phenylhydrazin  are  mixed  in  a  test-tube  with  10  drops  of  glacial 
acetic  acid  and  1  C.C.  of  a  saturated  solution  of  common  salt.  To 
this  are  added  :;  c.C.  of  urine,  when  the  mixture  is  boiled  for  two 
minutes  and  is  then  s,.(  aside  to  cool.  in  the  presence  of  more 
than  0.5  per  cent,  of  glucose,  crystals  of  phenyl-glucosazon  begin 

to  Separate    out  after  one  or  two   minute-.      Should    smaller  amounts 

be  present,   it  is  necessary  to  wait.     The  sedimenl   is  then  exam- 


280  THE   URINE. 

ined  microscopically.  As  we  are  generally  only  dealing  with  glu- 
cose in  the  urine,  a  further  examination  is  usually  not  necessary, 
especially  if  the  substance  crystallizes  out  in  large  needles,  which  are. 
often  collected  in  stars  and  sheaves.  To  identify  these  further,  how- 
ever, their  melting-point  must  be  determined.  As  has  been  stated, 
this  differs  in  the  different  osazons,  with  the  exception  of  lsevulose 
and  glucose,  which  have  the  same  melting-point.  Lsevulose,  how- 
ever, is  found  only  under  exceptional  conditions.  Its  presence  may 
be  established  as  shown  below.  The  melting-points  of  the  various 
osazons  which  may  be  encountered  are  as  follows : 

Glucose 204°-205°  C. 

Lsevulose 204°-205°  C. 

Galactose -  193°  C. 

Maltose 206°  C. 

Isomaltose       150°-153°  C. 

Lactose 200°  C. 

Arabinose 159°  C. 

Xvlose 159°  C. 

Glucuronic  acid 114°-115°  C. 

The  glucosazon  is  insoluble  in  water,  but  dissolves  with  ease  in 
hot  alcohol,  from  which  it  can  be  precipitated  on  cooling  in  crystal- 
line form,  by  diluting  with  water.  The  crystals  are  then  collected 
on  a  filter,  dried  over  sulphuric  acid,  and  further  examined  if 
desired. 

Polarimetric  Examination. — The  polarimetric  examination 
for  the  presence  of  sugar  should  always  be  controlled  by  one  or 
more  of  the  tests  that  have  just  been  described.  Dextrorotation, 
unless  biliary  acids  are  present,  can  be  directly  referred  to  the 
presence  of  sugar,  and  usually  to  glucose.  Lsevorotation,  however, 
may  be  referable  to  other  reducing  substances  besides  lsevulose, 
such  as  the  conjugate  glucuronates,  /3-oxy butyric  acid,  and  others. 
If  such  substances,  moreover,  are  present  in  larger  amounts,  traces 
of  dextrose  may  be  overlookod.  It  is  hence  advisable  to  examine 
the  urine  both  before  and  after  treatment  with  yeast,  and  in  doubt- 
ful cases  to  control  the  quantitative  results,  which  are  obtained  by 
the  polarimeter,  by  some  other  method.  For  a  detailed  description 
of  this  method  I  must  refer  the  reader  to  special  works.  In  every 
case  the  urine  must  be  perfectly  clear  and  free  from  albumin.  If 
highly  colored,  it  should  be  treated  with  lead  acetate  solution  and 
then  filtered,  in  which  case  allowance  must  be  made  for  the  degree 
of  dilution  if  quantitative  results  are  desired. 

Quantitative  Estimation. — Differential  Density  Method. — 
The  methods  available  for  the  quantitative  estimation  of  glucose 
are,  like  the  common  tests,  on  the  whole  most  unsatisfactory.  The 
least  objectionable  perhaps  is  that  based  upon  the  determination  of 
the  difference  in  the  specific  gravity  before  and  after  fermentation. 
It  has  been  found  that  a  diminution  by  0.001  corresponds  to  the 
previous  presence  of  0.230  per  cent,  of  sugar. 


THE  CARBOHYDRATES.  281 

The  specific  gravity  is  first  determined  in  the  fresh  urine,  after 
adding  2  grammes  of  tartrate  of  potassium  and  sodium,  and  2 
grammes  of  diacid  sodium  phosphate  to  every  100  c.c.  To  about 
200  c.c.  which  have  thus  been  prepared,  from  5  to  10  grammes 
of  fresh  yeast  are  added,  and  the  mixture  is  set  aside  at  a  tem- 
perature of  from  20°  to  25°  C.  until  fermentation  is  completed.  If 
but  little  sugar  is  present,  two  or  three  hours  will  suffice;  otherwise 
the  mixture  is  allowed  to  stand  for  twelve  hours.  Evaporation 
is  guarded  against  by  closing  the  bottle  with  a  perforated  stopper 
through  which  a  finely  drawn  out  tube  passes,  which  is  open  at  the 
distal  end.  The  specific  gravity  is  then  again  determined  at  the  same 
time  as  before,  and  the  difference  multiplied  by  0.230.  The  result 
indicates  the  amount  of  sugar  in  per  cent. 

The  method  yields  good  results  unless  very  small  amounts  of 
sugar  are  present,  viz.,  less  than  0.5  per  cent.  In  such  an  event, 
the  reducing  power  of  the  urine  is  first  ascertained  according  to 
Knapp's  method.  It  is  then  fermented,  when  the  remaining  re- 
ducing substances  are  again  determined.  The  difference  may  be 
referred  to  sugar. 

Fermentation  Method. — In  the  clinical  laboratory  especially 
constructed  saccharimetric  tubes  are  used,  of  which  Lohnstein's  is 
probably  the  best.  These  are  provided  with  a  scale  which  enables 
the  percentage  of  sugar  to  be  read  off  directly  from  the  amount  of 
carbonic  acid  that  has  gathered  in  the  upper  end  of  the  tube.  The 
instruments  are  accompanied  by  printed  instructions  for  use,  which 
need  not  be  considered  at  this  place. 

Kxapp's  Method. — This  method  is  to  be  preferred  to  the  older 
method  of  Fehling,  which  furnishes  results  of  value  only  in  espe- 
cially experienced  hands. 

The  method  is  based  upon  the  observation  that  mercuric  cyanide 
in  alkaline  solution  is  reduced  by  sugar  to  metallic  mercury.  If 
urine  is  then  added  to  a  solution  containing  a  known  amount  of  the 
cyanide  until  this  is  entirely  reduced,  the  corresponding  amount  of 
sugar  can  be  directly  ascertained. 

The  solution  which  is  generally  employed  for  this  purpose  con- 
tains 10  grammes  of  the  chemically  pure  cyanide,  and  100  c.c.  of  a 
solution  of  sodium  hydrate  (sp.  gr.  1.145)  in  the  liter:  20  c.c.  cor- 
respond to  0.05  gramme  of  glucose. 

\\v  urine  must  be  free  from  albumin  and  should  contain  not 
more  than  0.5  to  1  per  cent,  of  sugar.  This  should  first  be  ascer- 
tained by  a  preliminary  test.  If  more  is  present,  the  urine  should 
be  correspondingly  diluted. 

Twenty  e.e.  of  the  reagent  are  diluted  with  80  c.c.  of  distilled 
water,  or  with  less  if  ;i  smaller  amount  of  sugar  than  0.5  per  cent, 
i-  present.  The  solution  is  heated  to  the  boiling-point,  and  then 
titrated  with  the  diluted  urine,  boiling  for  one-half  minute  after  the 
addition  of  every  2  e.e.  or  Less  of  the  urine.  As  the  end-reaction 
is  approached,  the  mercury  together  with  the  phosphates  settles  to 


282  .  THE   URINE. 

the  bottom  and  the  supernatant  fluid  becomes  clear.  The  final  point 
is  reached  when  a  drop  of  the  liquid,  placed  upon  filter-paper,  and 
successively  held  over  the  mouth  of  a  bottle  containing  fuming 
hydrochloric  acid  and  over  that  of  one  containing  a  strong  solution 
of  hydrogen  sulphide,  is  no  longer  colored  yellow.  The  results  are 
then  calculated  on  the  basis  outlined  above. 

Fehling's  Method. — For  a  consideration  of  this  method,  which 
at  best  is  open  to  numerous  objections,  and  which  sometimes  leads  to 
no  end  whatever,  the  reader  is  referred  to  other  works.  At  this 
place  it  may  well  be  omitted. 

Lactose. — The  presence  of  lactose  in  the  urine  is  a  normal 
occurrence  in  nursing  women,  and  it  is  at  times  found  also  imme- 
diately preceding  confinement.  Its  appearance  in  the  urine  is  un- 
doubtedly referable  to  absorption,  owing  to  the  fact  that  a  super- 
abundance of  milk  is  being  produced,  and  we  accordingly  also  find 
the  substance  in  the  urine  when  for  any  reason  lactation  is  sup- 
pressed. Once  it  has  found  its  way  into  the  circulation,  its  elimina- 
tion through  the  kidneys  necessarily  follows,  as  the  body  is  incapable 
of  inverting  the  disaccharides  to  monosaccharides. 

Aside  from  its  occurrence  in  connection  with  lactation,  lactose 
is  found  in  the  urine  only  if  abnormally  large  amounts  have  been 
ingested.  In  such  an  event,  as  has  been  stated,  a  certain  proportion 
of  the  sugar  escapes  inversion  in  the  epithelial  cells  which  line  the 
intestinal  tract,  and  on  entering  the  general  circulation  it  is  elimi- 
nated as  such.  The  amount  of  lactose  which  may  be  found  in  the 
urine  of  nursing  women  varies  between  0.013  and  0.438  per  cent. 
Its  presence  in  the  urine  may  be  suspected  if  the  reduction  test  and 
the  phenylhydrazin  test  yield  a  positive  result,  while  the  fermenta- 
tion test,  as  usually  conducted,  is  negative.  Like  glucose,  the  sub- 
stance is  dextrorotatory.  To  identify  the  sugar  positively  as  lactose, 
however,  it  is  necessary  to  isolate  it  as  such. 

Isolation. — The  collected  urine  of  twenty-four  hours  is  precipi- 
tated with  lead  subacetate  and  filtered.  After  washing  with  water 
the  filtrate  and  washings  are  mixed  and  treated  with  ammonia. 
The  resulting  precipitate  is  filtered  off  and  the  filtrate  again  pre- 
cipitated with  lead  subacetate  and  ammonia,  and  so  on  until  the 
final  filtrate  is  optically  inactive.  The  precipitates,  with  the  excep- 
tion of  the  first,  are  then  mixed,  washed  with  water,  decomposed 
with  hydrogen  sulphide,  and  filtered.  In  the  filtrate  the  excess  of 
hydrogen  sulphide  is  removed  by  a  current  of  air,  and-  freed  from 
any  acids  that  have  been  liberated  by  shaking  with  argentic  oxide. 
The  mixture  is  filtered,  freed  from  soluble  silver  with  hydrogen  sul- 
phide, treated  with  barium  carbonate,  and  concentrated  to  a  small 
volume ;  90  per  cent,  alcohol  is  then  added,  which  causes  the 
formation  of  a  flocculent  precipitate.  This  is  filtered  off.  The 
filtrate  is  placed  in  the  desiccator,  when  on  standing  crystals  of 
lactose  gradually  separate  out.     These  may  be  purified  by  recrys- 


THE   CARBOHYDRATES.  283 

tallization,  decolorization  with  animal  charcoal,  and  extraction  with 
60-70  per  cent,  alcohol. 

Laevulose. — The  occurrence  of  a  laevorotatory  sugar  has  been  at 
times,  though  rarely,  observed  in  the  urine  of  diabetic  patients, 
where  it  was  present  either  alone  or  in  association  with  glucose. 
Like  dextrose,  the  substance  reduced  Fehling's  and  Nylander's  solu- 
tion, and  formed  an  osazon  with  phenylhydrazin,  with  a  melting- 
point  of  205°  C.  It  was  fermentable,  but,  unlike  true  laevulose, 
could  be  precipitated  with  basic  lead  acetate. 

Leo's  laiose,  which  was  also  obtained  from  a  diabetic  urine  on 
one  occasion,  was  very  similar  to  the  body  just  described,  but,  unlike 
this,  could  not  be  fermented.  With  phenylhydrazin,  moreover,  it 
formed  a  yellowish-brown  non-crystallizable  oil. 

Of  the  nature  of  these  bodies  nothing  further  is  known. 

The  presence  of  a  laevorotatory  sugar  can,  of  course,  readily  be 
established  by  the  common  tests,  supplemented  by  a  polarimetric 
examination,  if  it  is  present  alone.  If  glucose,  however,  also  is  con- 
tained in  the  urine  in  amounts  sufficient  to  counteract  the  laevorota- 
tion,  the  matter  is  more  difficult.  In  such  an  event,  however,  it  will 
be  observed  that  higher  values  are  obtained  in  estimating  the  sugar 
with  Knapp's  method,  or  according  to  the  differential  density  method, 
than  with  the  polarimeter,  for  reasons  which  are  self-evident. 

Maltose. — Maltose  together  with  glucose  was  found  on  one  occa- 
sion in  the  urine  of  a  patient  supposedly  the  subject  of  pancreatic 
disease.  Its  recognition  is  essentially  dependent  upon  the  forma- 
tion of  its  osazon,  and  the  identification  of  the  latter  by  its  melting- 
point  (see  also  page  59). 

Dextrin. — That  traces  of  dextrin  are  found  in  the  urine  under 
normal  conditions  has  been  pointed  out.  Larger  amounts  have  been 
observed  in  the  case  of  a  diabetic  patient,  where  the  substance 
apparently  took  the  place  of  glucose.  Of  its  origin  nothing  definite 
is  known,  but  it  is  likely  that  in  health  the  substance  gains  entrance 
to  the  circulation  in  a  more  or  less  accidental  way,  and  is  then, 
of  course,  eliminated  at  once.  It  is  now  regarded  as  identical  with 
the  animal  gum  of  Landwehr. 

To  demonstrate  the  presence  in  normal  urine  of  carbohydrates  of 
this  character,  the  urine  is  boiled  witli  dilute  sulphuric  acid  for  about 
thirty  minutes,  and  after  being  rendered  alkaline  with  sodium  hydrate 
is  examined  with  Nylander's  lest.  A  positive  reaction  may  now  be 
obtained,  while  previously  no  reduction  occurred. 

For  the  isolation  of  these  normal  carbohydrates  the  reader  is 
referred  to  special   works. 

Pentoses. — Thai  traces  of  pentoses  may  occur  in  the  urine  under 
normal  conditions  has  been  stated.  They  are  due  then,  no  doubt, 
to  the  ingestion  of  such  articles  of  food  as  prunes,  cherries,  grapes, 
beer,  wine,  etc.  It  will  be  shown,  ii loreo ver,  t hat  the  peculiar  gluco- 
proteid  which  occur-  in  the  pancreas  yields  a  pentose  on  decom- 
position, and    it    i-    possible   (hat    this  also  may  at  times  be    a   source 


284  THE   URINE. 

of  the  pentoses  which  are  found  in  the  urine.  As  a  general  rule, 
it  is  true,  ingested  pentoses  are  mostly  decomposed  within  the  body ; 
but  in  chickens  and  rabbits,  in  which  a  more  marked  ability  exists 
to  effect  their  oxidation  than  in  man,  this  is  never  complete.  As  in 
the  case  of  glucose,  the  power  to  assimilate  pentoses  seems  to  vary 
with  different  individuals,  and  here,  as  there,  a  digestive  pentosuria 
can  artificially  be  produced.  In  diabetes  also  the  power  to  oxidize 
the  pentoses  may  be  much  impaired,  and,  very  curiously,  the  largest 
quantities  have  thus  far  been  observed  in  morphin  habitues. 

The  individual  pentoses  which  have  thus  far  been  encountered  in 
the  urine  are  arabinose,  xylose,  and  rhamnose.  They  all  reduce 
Fehling's  solution,  and  give  rise  to  osazons  with  phenylhydrazin. 
The  melting-points  of  the  resulting  compounds,  however,  are  differ- 
ent from  those  of  the  common  hexosazons  (see  page  280).  In  the 
amounts,  however,  in  which  they  are  usually  present  no  reactions 
are  obtained  in  this  manner.  The  fermentation  test  is  not  obtained. 
Xylose  and  rhamnose  turn  the  plane  of  polarization  to  the  right, 
while  arabinose  is  optically  inactive. 

To  demonstrate  the  presence  of  pentoses  in  the  urine  the  following 
test  of  Tollens  is  employed  : 

A  saturated  solution  of  orcin  in  concentrated  hydrochloric  acid  is 
first  prepared,  and  should  contain  a  slight  excess  of  the  substance. 
Six  c.c.  of  this  are  divided  into  two  equal  parts  and  are  allowed  to 
cool.  To  one  portion  0.5  c.c.  of  the  urine  under  examination  is 
added,  while  the  other  is  treated  with  the  same  amount  of  normal 
urine  of  a  like  specific  gravity.  In  either  case  it  is  well  first  to 
decolorize  the  urine  with  animal  charcoal.  Both  specimens  are 
then  placed  in  a  beaker  with  boiling  water,  when  the  urine  contain- 
ing pentoses  gradually  assumes  a  green  color,  which  begins  in  the 
surface  layers,  while  the  color  of  the  normal  urine  is  scarcely  changed. 
One-tenth  per  cent,  of  pentoses  can  thus  be  demonstrated. 

With  Tollens'  phloroglucin  test,  which  is  conducted  in  the  same 
manner,  a  deep-red  color  develops  instead ;  but  this  reaction  is  also 
common  to  the  glucuronates.  (The  reagent  is  prepared  in  the  same 
manner  as  in  the  case  of  the  orcin  reagent,  phloroglucin  being  simply 
substituted  for  the  orcin.) 

THE  ALBUMINS. 

As  every  urine  contains  a  small  number  of  cellular  elements 
which  are  derived  from  the  urinary  tract,  it  can  readily  be  under- 
stood that  even  under  normal  conditions  albumin  can  be  demon- 
strated with  suitable  methods.  The  amount,  however,  is  exceed- 
ingly small,  and  with  the  common  tests  a  positive  reaction  cannot 
be  obtained  unless  the  substance  in  question  has  been  previously 
isolated  from  a  large  quantity  of  urine.  In  such  an  event  a  trace 
of  a  nucleo-albumin  can  be  demonstrated.  The  occurrence  of  the 
common  albumins  of  the  blood,  on   the  other  hand,   is  always  a 


THE  ALBUM  IS  S.  285 

pathological  phenomenon.  Some  writers,  it  is  true,  speak  of  a 
physiological  albuminuria,  which  may  be  observed  after  severe 
muscular  exercise,  following  cold  baths,  during  pregnancy,  etc., 
and  it  is  even  claimed  that  the  elimination  of  albumin  in  young 
persons,  which  is  so  commonly  observed  in  neurotic  and  anremic 
individuals,  in  association  with  an  increased  amount  of  uric  acid 
and  oxalic  acid,  belongs  to  this  order.  There  is  an  increasing 
tendency  among  pathologists,  however,  to  doubt  the  physiological 
character  of  such  forms  of  albuminuria,  and  personally  I  main- 
tain that  every  albuminuria  of  a  hajmatogenic  type  is  a  pathological 
phenomenon/  We  may,  in  fact,  go  further,  and  assume  that  the 
appearance  of  nucleo-albumin  also  in  amounts  which  can  be  de- 
monstrated by  ordinary  tests  is  abnormal,  as  such  an  occurrence 
must  of  necessity  be  associated  with  an  increased  desquamation  of 
epithelial  cells,  which  in  itself  is  evidence  of  a  pathological  process. 
This,  however,  is  hardly  the  place  to  enter  upon  a  detailed  con- 
>ideration  of  the  various  morbid  conditions  in  which  albuminuria 
may  occur,  and  it  will  suffice  to  state  that  any  disturbance  in  the 
nutrition  of  the  glandular  elements  of  the  kidney,  from  whatever 
cause,  will  at  once  find  expression  in  the  appearance  of  albumin  in 
the  urine.  The  albumins  which  are  then  eliminated  are  the  common 
albumins  of  the  blood,  and  notably  serum-albumin  and  serum- 
globulin.  Fibrinogen,  on  the  other  hand,  is  usually  not  found.  In 
cases  of  hematuria  and  chyluria,  however,  its  presence  may  be 
inferred  from  the  formation  of  coagula  of  fibrin.  This  may  occur 
in  the  urinary  passages  already,  but  more  commonly  it  is  observed 
after  the  urine  has  been  voided. 

Other  albumins  besides  those  which  are  normally  found  in  the 
blood  are  encountered  only  exceptionally  in  the  urine  ;  but  it  may 
be  stated  that  whenever  such  substances  find  their  way  into  the 
general  circulation  their  elimination  at  once  follows.  Formerly 
it  was  taught  that  peptones  could  thus  appear,  and  for  many  years 
various  types  of  peptonuria  were  described.  More  recent  investiga- 
tion-, however,  have  shown  that  the  substances  in  question  were  in 
realitv  no  peptones  in  the  sense  of  Kiilme,  but  albumoses.  Some 
of  these  are,  no  doubt,  identical  with  the  common  digestive  albu- 
moses, and  find  their  way  into  the  blood,  when  their  further  trans- 
formation into  native  albumins  does  not  occur  in  the  epithelial 
ce|]s  ,,f  the  digestive  tract.  Others  again  are  probably  formed  in 
the  body  proper  in  diseases  which  are  associated  with  suppurative 
processes,  and  in  which  the  formation  of  albumoses  occurs  at  the 
expense  of  the  tissue  albumins  under  the  influence  of  various  micro- 
organisms, ruder  still  other  conditions,  as  in  the  various  non- 
septic  fevers,  in  phosphorus  poisoning,  etc.,  the  albumosuria  may 
We  the  expression  of  ;i  metabolic  abnormality  {><■/■  se,  and  is  possibly 
dependent  upon  the  action  of  the  various  tissue  ferments. 

Of  special  interest,  further,  i-  the  appearance  in  the  urine  of  the 
so-called   albumin  of  Bence  done-,  which   has  been   repeatedly  ob- 


286  THE   URINE. 

served  in  association  with  multiple  myelomata  of  the  bones.  Of  its 
chemical  nature,  however,  little  is  known.  By  some  it  is  regarded 
as  an  albumose,  but,  according  to  Neumeister,  it  is  not  identical 
with  any  of  the  known  digestive  albumoses.  I  shall  revert  to  it 
later. 

In  diseases,  finally,  in  which  an  increased  destruction  of  leuco- 
cytes is  taking  place,  both  histon  and  nucleohiston  have  been  found. 

Of  other  albumins  which  are  foreign  to  the  blood,  only  egg- 
albumin  has  been  encountered,  following  the  ingestion  of  excessive 
amounts  of  the  substance. 

Tests  for  the  Common  Albumins  of  the  Blood. — The  Niteic 
Acid  Test. — A  small  amount  of  urine  is  placed  in  a  conical  glass 
and  is  underlaid  with  a  few  cubic  centimeters  of  concentrated  nitric 
acid,  when  in  the  presence  of  serum-albumin  and  serum-globulin  a 
white,  opaque  disk  of  coagulated  albumin  is  formed  at  the  zone  of  con- 
tact, which  varies  in  intensity  and  extent  with  the  amount  of  albumin 
present.  Immediately  below  this  variously  colored  rings  also  are 
observed,  which  are  in  part  referable  to  the  decomposition  of 
indoxyl  and  skatoxyl  sulphate,  and  the  oxidation  of  the  liberated 
indoxyl  and  skatoxyl  to  blue  and  red  pigments.  In  the  presence 
of  bile-pigment  a  green  color  will  then  also  be  noted.  If  much 
urea  be  present  at  the  same  time,  it  may  happen  that  after  a  few 
minutes  a  dense  disk  of  urea  nitrate  crystals  separates  out  in  the 
lower  pigmented  layer,  but  more  commonly  these  are  formed 
throughout  the  mixture  on  standing,  and  gradually  settle  to  the 
bottom.  Should  uric  acid,  further,  be  present  in  increased  amount, 
a  white  disk  develops  higher  up  in  the  urine,  and  separated  from 
that  referable  to  albumins  by  a  layer  of  clear  urine.  This  may  at 
times  be  quite  marked,  and  may  extend  downward  toward  the  nitric 
acid  so  rapidly  that  it  is  difficult  to  say  whether  it  is  referable  to 
albumin  or  a  large  excess  of  uric  acid.  Should  this  occur,  it  is  best 
to  dilute  the  urine  with  an  equal  volume  of  water,  or,  even  more 
strongly,  when  a  portion  of  the  uric  acid  at  least  is  prevented  from 
separating  out  or  is  held  in  solution  altogether. 

As  nucleo-albumins,  when  present  beyond  traces,  can  simulate 
the  true  albumin  reaction,  it  is  well  to  dilute  the  urine  with  water 
and  to  examine  again  when  its  presence  is  suspected.  If  then  the 
reaction  is  more  pronounced  than  before,  the  precipitate  may,  in  part 
at  least,  be  referable  to  this  source.  This  possibility  should  be  con- 
sidered if  the  urine  contains  an  increased  number  of  morphological 
elements,  and  if  the  reaction  is  slight.  Other  tests  should  then  also 
be  employed. 

Albumoses,  if  present  beyond  traces,  also  react  with  nitric  acid, 
but  it  is  to  be  noted  that  in  such  cases  the  precipitate  disappears  on 
heating  and  reappears  on  cooling,  while  the  liquid  at  the  same  time 
assumes  an  intensely  yellow  color.  Should  a  mixed  albuminuria 
exist — i.  e.,  should  albumoses  and  albumin  be  present  simultane- 
ously— the  clearing  of  the  urine  is  only  partial. 


THE  ALBUM IX S.  287 

As  nitric  acid  also  precipitates  certain  resins  which  may  have 
been  administered  for  medicinal  purposes,  it  is  at  times  necessary  to 
eliminate  this  possibility  of  error.  Their  presence  is  indicated  if 
the  precipitate  disappears  on  shaking  the  mixture  with  ether. 

The  Boiling  Test. — The  urine  should  present  a  feebly  acid  or 
neutral  reaction.  If  alkaline,  it  is  rendered  nearly  neutral  by  adding 
a  drop  or  two  <»f  dilute  acetic  acid.  A  few  cubic  centimeters  are 
then  boiled  in  a  test-tube,  when  in  the  presence  of  coagulable 
albumins  the  liquid  becomes  turbid,  and  on  standing  a  flocculent 
precipitate  gathers  at  the  bottom  of  the  tube.  The  turbidity  may, 
however,  at  times  be  due  to  a  precipitation  of  neutral  earthy  phos- 
phates. To  distinguish  between  the  two,  one  or  two  drops  of  a  25 
per  cent,  solution  of  nitric  acid  are  now  added  for  every  1  c.c.  of 
the  urine.  The  earthy  phosphates  are  thus  dissolved,  while  the  pre- 
cipitate of  albumin  remains  unaffected.  If  more  than  traces  of 
albumin  are  present,  this  test  is  very  reliable  ;  otherwise  there  is 
danger  of  dissolving  the  small  amount  of  albumin.  If  nitric  acid 
is  used  instead  of  acetic  acid,  this  danger  is  generally  small,  how- 
ever, but  the  possibility  exists,  nevertheless.  Hence  in  doubtful 
cases  it  is  always  best  to  resort  to  the  nitric  acid  test  as  well. 

If  the  albumin  of  Bence  Jones  should  be  present,  coagulation 
occurs  at  a  temperature  of  50°  C.  already,  but  it  will  be  noted 
that  the  precipitate  disappears  on  subsequent  boiling  and  reappears 
on  cooling. 

The  common  albumoses,  as  well  as  nucleo-albumin,  are  not 
thrown  down.  The  presence  of  the  former  may  be  inferred  if 
after  the  addition  of  the  acid  and  subsequent  cooling  a  white  pre- 
cipitate is  formed,  which  dissolves  upon  the  application  of  heat  and 
reappears  on  cooling. 

If  acetic  acid  is  to  be  employed  instead  of  nitric  acid,  it  is  best 
to  treat  the  urine  with  one-sixth  of  its  volume  of  a  saturated  solu- 
tion of  common  salt,  after  having  rendered  it  distinctly  acid.  It 
is  then  boiled  as  before.  In  this  case  the  danger  of  dissolving  the 
precipitated  albumins  is  much  lessened. 

The  Potassium  Ferrocyanide  Test. — A  few  cubic  centimeters  of 
urine  are  strongly  acidified  with  acetic  acid,  and  treated  with  a 
10  per  cent,  solution  of  potassium  ferrocyanide  drop  by  drop,  when 
in  the  presence  of  albumin  a  precipitation  occurs  which  varies 
in  intensity  with  the  amount  present.  Concentrated  urines  should 
first  be  diluted.  Albumoses  are  also  thrown  down,  but  are  redis- 
solved  on  boiling  and  reappear  on  cooling.  Should  nucleo-albu- 
mins  be  present  beyond  traces,  a  precipitate  develops  upon  the 
addition  of  the  acetic  acid.  This  may  also  occur  if  urates  are 
pre-,. nt  in  large  amounts.  But  in  this  event  the  precipitate  clears 
upon  warming  the  solution  ;  and  if  the  urine,  moreover,  is  previously 
diluted,  ii  doe-;  not  occur  at  all,  while  the  separation  of  nucleo-albu- 
min takes  place  more  rapidly  in  the  latter  ease  than  before. 

Still    other   teste  exisl  which  are  equally  good,  but   for  practical 


288  THE   URINE. 

purposes  the  three  just  described  will  suffice.  In  every  case, 
however,  it  is  necessary  that  the  urine  should  be  perfectly  clear. 
If  turbid,  owing  to  precipitation  of  any  of  the  normal  constit- 
uents of  the  urine,  simple  filtration  generally  suffices  ;  but  if  refer- 
able to  bacteria,  it  is  best  shaken  with  powdered  talcum  and  then 
filtered. 

Special  Test  for  Serum-albumin. — If  it  is  desired  to  demonstrate 
the.  presence  of  serum-albumin  by  itself,  the  urine  is  rendered 
amphoteric  or  faintly  alkaline  with  sodium  hydrate,  and  satu- 
rated with  magnesium  sulphate  to  remove  any  globulins  that  may 
be  present.  The  filtrate  is  then  acidified  with  acetic  acid  and  boiled, 
when  in  the  presence  of  serum-albumin  precipitation  occurs. 

Special  Test  for  Serum-globulin. — This  is  conducted  as  above, 
or  by  adding  an  equal  volume  of  a  saturated  solution  of  ammo- 
nium sulphate  to  the  amphoteric  urine,  when  the  globulins  are 
thrown  down.  Urates  may  then  also  separate  out,  but  this  always 
occurs  later.  The  precipitated  globulins  are  soluble  in  acetic  acid. 
As  I  have  stated  before,  there  is  one  instance  of  globulinuria  on 
record  in  which  the  substance  was  found  in  the  sediment  in  crystal- 
line form. 

Test  for  Nucleo-albumin. — A  small  amount  of  urine  is  diluted 
with  water  and  then  treated  drop  by  drop  with  strong  acetic  acid. 
In  this  manner  the  precipitation  of  urates  is  prevented,  while  the 
nucleo-albumin  separates  out.  To  identify  it  as  such,  however,  it  is 
necessary  to  isolate  the  substance  in  larger  amounts.  To  this  end, 
the  collected  urine  of  twenty-four  hours  is  carefully  neutralized  and 
concentrated  to  about  1000  c.c.  at  a  temperature  of  60°-70°  C.  On 
filtering,  it  is  saturated  with  ammonium  sulphate  in  substance  and 
the  precipitate  collected  on  a  filter.  This  is  dissolved  in  a  little 
water  and  freed  from  salts  by  dialysis.  Should  hetero-albumosebe 
present,  this  separates  out  and  is  removed  by  filtration.  A  portion 
of  the  remaining  solution  is  then  tested  with  acetic  acid,  as  described  ; 
the  precipitate  should  be  soluble  in  mineral  acids.  In  the  remaining 
solution  the  body  is  completely  thrown  down  with  acetic  acid.  The 
precipitate  is  filtered  off,  washed  with  dilute  acetic  acid,  and  dried. 
On  fusion  with  caustic  alkali  and  potassium  nitrate,  phosphoric  acid 
should  then  be  liberated  if  the  substance  is  a  nucleo-albumim  If 
this  reaction  is  not  obtained,  but  if  on  boiling  with  dilute  mineral 
acids  a  reducing  substance  is  set  free,  it  may  be  assumed  that  the 
body  in  question  is  mucin. 

Test  for  Albumoses. — To  test  for  albumoses  in  general,  a  small 
amount  of  the  urine  is  acidified  with  acetic  acid  and  treated  with  an 
equal  volume  of  a  saturated  solution  of  common  salt.  The  solution 
is  then  boiled  and  filtered  while  still  hot,  so  as  to  remove  any  eoagu- 
lable  albumins  that  may  be  present.  On  cooling,  the  albumoses 
separate  out,  but  redissolve  on  boiling.  In  such  an  event,  the  solu- 
tion also  gives  the  biuret  reaction  and  that  of  Millon. 

Safer,  however,  is  the  following  method,  which  should  always  be 


THE  ALBUMINS.  289 

employed  when  there  is  reason  to  believe  that  albumoses  are  present 
only  in  traces.  The  collected  twenty-four  hours'  urine  is  carefully 
neutralized,  concentrated  to  about  1000  c.c.  at  60°-70°  C,  filtered, 
and  saturated  with  ammonium  sulphate  in  substance.  The  pre- 
cipitate is  collected  on  a  filter,  and  dissolved  in  a  little  water,  when 
a  small  portion  is  treated  with  an  equal  volume  of  a  saturated  solu- 
tion of  common  salt,  and  with  acetic  acid  or  nitric  acid  drop  by 
drop  so  long  as  any  precipitate  that  has  formed  is  thus  increased. 
The  solution  is  then  boiled.  If  coagulable  albumins  are  present, 
these  are  precipitated,  and  are  filtered  off  from  the  hot  solution.  If 
the  filtrate  becomes  turbid  again  on  cooling,  and  clears  upon  sub- 
sequent boiling,  the  presence  of  albumoses  may  be  inferred.  To 
determine  the  character  of  the  albumoses  in  question,  the  remaining 
liquid  is  dialyzed  (see  above),  freed  from  nucleo-albumin  by  means 
of  acetic  acid,  neutralize,!,  concentrated  on  a  water-bath,  and 
saturated  with  rock-salt.  If  primary  albumoses  are  present,  they 
are  thus  precipitated  and  filtered  off.  When  acetic  acid  that  has 
been  saturated  with  common  salt  is  added  to  the  filtrate  the  deutero- 
albumoses  are  thrown  down.  This  test  should  be  applied  in  the 
preliminary  examination  also  if  no  reaction  is  obtained. 

Instead  of  using  sodium  chloride  to  precipitate  the  albumoses, 
ammonium  sulphate  can,  of  course,  also  be  used,  as  has  been 
described  on  page  184. 

True  peptones,  in  the  sense  of  Kiihne,  do  not  occur  in  the 
urine,  and  it  is  hence  unnecessary  to  describe  the  older  and  more 
complicated  methods  which  formerly  were  employed  in  their  search. 

Bence  Jones'  Albumin. — This  body,  as  has  been  pointed  out,  has 
repeatedly  been  encountered  in  the  urine  in  association  with  the 
existence  of  multiple  myelomata  of  the  bones.  Of  its  nature, 
however,  little  is  known.  Most  observers  have  regarded  it  as  an 
albumose,  but  it  is  admitted  that  it  is  not  identical  with  any  of  the 
known  digestive  albumoses.  Like  the  globulin  described  bv  Paton, 
it  has  been  found  in  crystalline  form  in  the  urinarv  sediment. 
Magnus- Levy,  who  has  recently  studied  the  bodv  in  question,  while 
likewise  unable  to  identify  it  with  any  of  the  known  albumins  or 
albumoses,  points  out  that  it  has  in  realitv  only  one  propertv  in 
common  with  the  albumoses,  viz.,  the  solubility  of  its  precipitate  on 
boiling.  Be  points  out,  however,  that  this  is  only  apparent,  and 
that  under  suitable  conditions  the  body  is  coagulated  on  heating  to 
100°  C,  like  the  native  albumins.  He  further  noted  that,  like  the 
true  albumins,  the  substance  yields  the  common  digestive  products 
of  these  bodies,  viz.,  primary  and  secondary  albumoses;  but,  as  in 
the  case  of  casein,  no  hetero-group  could  be  demonstrated.  These 
results  T  have  personally  confirmed,  and  it  is  thus  conclusively 
iblished  thai  the  body  cannot  be  an  albumose.  Pending  further 
investigations,  it  is  hence  advisable  to  term  the  substance  the 
albumin  of  Bence  Jones.  Of  its  origin  nothing  definite  is  known. 
The  amount  whieh  i-  often  found,  however,  is  so  large  that  the  con- 

19 


290  THE    URINE. 

elusion  suggests  itself  that  the  substance  may  be  derived  from  the 
ingested  albumins,  and  is  formed  in  the  intestinal  canal  or  its 
walls  as  a  result  of  some  abnormal  digestive  process.  The  pres- 
ence of  the  albumin  may  be  suspected  if  a  urine  gives  the  usual 
albumose  reaction  to  a  marked  degree,  as  the  disease  in  question  is 
in  reality  the  only  one  in  which  larger  amounts  of  an  "  albumose"- 
like  body  are  obtained.  It  can  then  be  isolated  by  treating  the 
neutralized  urine  with  double  its  volume  of  a  saturated  solution  of 
ammonium  sulphate.  To  identify  the  substance,  it  is  advisable  to 
digest  the  body  with  pepsin,  to  demonstrate  the  formation  of  proto- 
albumose,  and  to  show  that  no  hetero-albumose  is  produced.  For 
further  details  the  reader  is  referred  to  Magnus-Levy's  work. 

Test  foe  Fibriist. — When  fibrin  is  present  in  the  urine  it  usually 
occurs  in  the  form  of  distinct  clots,  the  nature  of  which  is  commonly 
apparent  without  chemical  examination.  If  it  is  to  be  identified  in 
this  manner,  however,  the  clots  are  washed  with  water  until  free 
from  blood-pigments.  They  are  then  placed  in  a  5  per  cent,  solu- 
tion of  sodium  chloride  containing  an  excess  of  thymol,  to  guard 
against  putrefactive  changes.  It  will  be  observed  that  the  substance 
does  not  dissolve,  while  in  a  0.3  per  cent,  solution  of  hydrochloric 
acid  it  rapidly  swells  and  is  digested  after  the  addition  of  a  little 
pepsin. 

Test  for  Histon. — The  twenty-four  hours'  urine  is  first  freed 
from  coagulable  albumins  by  boiling.  It  is  then  precipitated  with 
a  large  excess  of  94  per  cent,  alcohol.  The  precipitate  is  washed 
with  hot  alcohol  and  dissolved  in  boiling  water.  On  cooling,  the 
solution  is  acidified  with  hydrochloric  acid  and  allowed  to  stand  for 
a  number  of  hours.  Any  uric  acid  that  has  separated  out  is 
removed  by  filtration,  when  the  filtrate  is  precipitated  with  ammonia. 
The  collected  material  is  washed  with  ammoniacal  water  until 
the  washings  no  longer  give  the  biuret  reaction.  It  is  then  dissolved 
in  dilute  acetic  acid.  If  histon  is  present,  the  solution  coagulates  on 
boiling  and  gives  the  biuret  reaction.  The  coagulated  material  dis- 
solves in  mineral  acids. 

Quantitative  Estimation  of  the  Coagulable  Albumins. — In 
the  clinical  laboratory  the  so-called  albuminimeters  of  Esbach  are 
conveniently  employed  for  this  purpose.  The  method  is  exceedingly 
simple,  and  gives  results  which  are  sufficiently  accurate  for  ordinary 
purposes.  To  this  end,  the  tube  is  filled  with  urine  to  the  mark  U. 
Esbach's  reagent,  which  consists  of  an  aqueous  solution  containing 
10  grammes  of  picric  acid  and  20  grammes  of  citric  acid  to  the 
liter,  is  then  added  to  the  mark  R.  The  tube  is  closed,  inverted  a 
number  of  times,  and  set  aside  for  twenty-four  hours.  The  number 
of  the  scale  which  corresponds  to  the  height  of  the  precipitated 
albumins  indicates  the  amount  in  grammes  in  1000  c.c.  of  urine. 
Care  must  be  had,  however,  that  the  urine  is  acid,  that  the  density 
does  not  exceed  1.006-1.008,  and  that  the  temperature  remains  at 
about  15°  C. 


THE  PIGMENTS   OF  THE    URINE.  291 

If  more  accurate  results  are  desired,  a  known  volume  of  urine, 
feebly  acidified  with  nitric  acid  if  necessary,  is  heated,  first  on  a 
water-bath  and  then  over  a  free  flame,  until  coagulation  is  com- 
plete. The  precipitate  is  eollected  on  a  small  filter,  and  washed 
with  water,  alcohol,  and  ether.  The  contained  nitrogen  is  now  esti- 
mated according  to  Kjeldahl's  method,  when  the  result  multiplied 
by  6.3  will  indicate  the  corresponding  amount  of  albumin. 

If  it  is  desired  to  estimate  the  amount  of  the  individual  albumins 
separately,  they  are  first  isolated,  as  has  been  described,  and  are  then 
subjected  to  Kjeldahl's  process.  In  this  case,  however,  ammonium 
sulphate  cannot  be  used  for  purposes  of  salting. 

THE  PIGMENTS  OF  THE  URINE. 

Of  the  chemical  nature  of  the  pigments  of  normal  urine  little  is 
known  that  is  definite.  According  to  some  observers,  the  yellow 
color  is  due,  in  part  at  least,  to  the  presence  of  so-called  urochrome, 
which  in  turn  is  regarded  as  identical  with  the  normal  urobilin  of 
MacMunn.  Others,  again,  claim  that  there  is  no  reason  to  .- u ]  > j  >■ . - . 
that  a  difference  exists  between  this  normal  urobilin  and  the  urobilin 
of  Jaffe,  which  is  mostly  observed  under  pathological  conditions,  but 
which  may  occur  also  in  health.  Jaffe's  urobilin,  further,  is  held  by 
some  to  be  identical  with  the  hydrobilirubin  which  results  from 
bilirubin  through  the  action  of  sodium  amalgam.  Of  late,  how- 
ever, this  view  has  been  questioned,  especially  as  bilirubin  on  oxida- 
tion furnishes  a  substance,  choletelin,  which  cannot  be  distinguished 
from  hydrobilirubin  on  the  one  hand,  or  urobilin  on  the  other.  A 
similar  pigment,  or  one  which  is  identical  with  urobilin,  has  further 
been  obtained  from  hsematoporphyrin.  That  the  urobilin  which  is 
notably  observed  under  pathological  conditions  can  lie  formed  within 
the  body  in  the  absence  of  micro-organisms  is  now  a  well-established 
feet.  We  thus  find  that  in  diseases  in  which  the  elimination  of  bile 
through  the  usual  channels  is  prevented,  urobilin  may  occur  in  the 
urine,  nevertheless;  and  it  has  further  been  noted  that  both  at  the 
beginning  and  at  the  end  of  jaundice  increased  amount-;  are  found. 
Similar  results  have  been  obtained  when  from  any  cause  an  increased 
destruction  of  blood-pigmeni  occurs.  We  may  thus  imagine  that  in 
such  cases  the  urobilin  results  from  bilirubin  through  an  extensive 
oxidation  to  choletelin.  This  view  of  the  origin  of  urobilin,  of 
'•our-'',  does  not  necessarily  preclude  the  possibility  that  a  certain 
amount  of  the  pigment,  which,  as  I  have  said,  may  normally  also 
occur  in  tin-  urine,  may  be  derived  from  bilirubin  through  a  process 
of  reduction  in  the  intestinal  tract.  But,  as  is  apparent  from  the 
considerations  jusl  related,  we  are  scarcely  in  a  position  to  speak 
authoritatively  of  the  origin  of  the  normal  urinary  pigments.  The 
chemical  position  of  the  colorless  mother-substance  of  urobilin,  more- 
over, which  i-  spoken  of  n-  urobilinogen,  and  which  can  usually  be 
demonstrated  whenever  urobilin  also  is  present, is  thufi  far  not  clear. 


292  THE   URINE. 

According  to  Garrod,  the  urobilin  of  the  urine  is  identical  with 
the  stercobilin  of  the  feces,  both  in  composition  and  properties,  but 
differs  conspicuously  from  hydrobilirubin,  especially  in  the  much 
smaller  percentage  of  nitrogen  which  it  contains,  viz.,  4.11  per  cent., 
as  compared  with  9.22.  The  elementary  composition  of  urobilin  is 
given  by  Garrod  and  Hopkins  as  :  C,  63.58  per  cent. ;  H,  7.84 ; 
N,  4.11,  and  O.  24.47.  Garrod  further  states  that  by  acting  upon 
urochrome  with  acids  he  never  succeeded  in  obtaining  any  prod- 
uct showing  the  urobilin  band,  or  yielding  the  well-known  fluores- 
cence with  zinc  chloride  and  ammonia,  as  Thudichum.  claimed. 
But  he  found  that  a  substance  having  both  these  properties  is 
readily  obtained  by  the  action  of  aldehyde  upon  an  alcoholic  solu- 
tion of  urochrome.  In  a  short  time — shorter  still  when  the  liquid 
is  warmed — an  absorption-band  appears  like  that  of  urobilin,  and 
the  tint  of  the  solution  deepens  to  a  rich  orange-yellow*  With  zinc 
chloride  and  ammonia  a  brilliant  green  fluorescence  occurs,  and 
the  band  is  shifted  toward  red,  as  that  of  urobilin  is  under  like 
conditions. 

Isolation  of  Urochrome. — To  demonstrate  the  presence  of  so-called 
urochrome  in  normal  urine,  the  fluid  is  acidulated  with  1  or  2  c.c. 
of  dilute  sulphuric  acid  pro  liter.  On  filtering,  it  is  saturated  with 
ammonium  sulphate.  The  resulting  precipitate  is  dried  and  ex- 
tracted with  warm  and  slightly  ammoniacal  alcohol.  The  pigment 
passes  into  solution,  and  is  obtained  on  evaporation  of  the  alcohol  as 
an  amorphous  reddish-brown  substance,  which  is  readily  soluble  in 
acidulated  water,  chloroform,  and  common  alcohol,  but  is  practically 
insoluble  in  ether  and  benzol.  According  to  Garrod,  its  solutions 
do  not  give  rise  to  any  bands  of  absorption,  and  do  not  fluoresce 
upon  the  addition  of  ammonia  and  zinc  chloride.  Gautier,  on  the 
other  hand,  states  that  its  acidulated  solutions  show  a  band  of 
absorption  about  F,  and  that  the  remainder  of  the  spectrum  from 
about  G  on  to  the  right  is  obscured.  He  adds  that  in  this  respect 
urochrome  and  choletelin  are  alike. 

Garrod  regards  the  action  of  aldehyde  upon  an  alcoholic  solution  of 
urochrome,  outlined  above,  as  a  very  delicate  test  for  the  pigment. 
The  process  can  be  stopped  then  by  simple  dilution  with  water,  as 
aldehyde  has  no  such  action  upon  aqueous  solutions  of  urochrome. 
If,  however,  the  action  is  allowed  to  continue,  a  further  change 
ensues.  The  liquid  reddens  and  a  second  band  appears  in  the  violet. 
The  fluorescence  can  still  be  obtained  with  zinc  chloride  and  ammonia, 
and  both  bands  are  shifted  toward  red  and  are  closer  together  than 
before. 

The  term  uroerythrin  has  been  applied  to  the  pigment  which 
imparts  the  salmon-red  color  to  sediments  which  are  composed  of 
urates  or  uric  acid.  Of  its  chemical  nature,  however,  nothing  defi- 
nite is  known,  but  there  is  evidence  to  show  that  it  also  is  a  deriva- 
tive of  the  normal  coloring-matter  of  the  blood.  It  contains 
62.51   per  cent,  of  carbon  and    5.79    per  cent,  of   hydrogen.     Its 


THE  PIGMENTS  OF  THE   URINE.  2lJ3 

amount  is  noticeably  increased  in  extensive  disease  of  the  liver,  as 
also  in  conditions  associated  with  an  increased  destruction  of  red 
corpuscles. 

Isolation  of  Uroerythrin. — As  has  been  stated,  the  salmon  color  of 
sediments  of  urates  and  uric  acid  is  due  to  this  pigment.  In  their 
absence  the  urine  is  precipitated  with  neutral  acetate  of  lead  or 
barium  chloride.  If  uroerythrin  is  present  beyond  traces,  it  is  thrown 
down,  and  colors  the  resulting  precipitate  a  more  or  less  intense 
salmon  red.  The  pigment  is  soluble  in  boiling  alcohol,  and  may 
thus  be  extracted.  Its  solutions  are  said  to  give  rise  to  two  bands 
of  absorption  to  the  left  of  F. 

Urobilin. — Urines  which  contain  much  urobilin,  viz.,  the  patho- 
logical  urobilin  of  Jaffe,  present  a  dark-yellow  color,  which  may  be 
imparted  to  the  foam  on  shaking.  They  are  thus  quite  similar  to 
icteric  urines. 

Tests. — To  identify  the  substance,  the  urine  is  precipitated  with  a 
mixture  of  barium  hydrate  and  barium  chloride.  If  notable  quanti- 
ties of  urobilin  are  present,  the  precipitate  is  thus  colored  a  more  or 
less  intense  brownish-red.  On  boiling  with  acidulated  alcohol  the 
pigment  is  then  extracted,  and  imparts  a  brownish  or  pomegranate- 
red  color  to  the  alcoholic  solution  (v.  Jaksch). 

Gerhardt's  test  also  is  very  serviceable.  To  this  end,  10-20  c.c. 
of  urine  are  extracted  with  chloroform  by  shaking.  A  few  drops 
of  a  dilute  solution  of  iodopotassic  iodide  are  added  to  the  extract, 
when,  upon  the  further  addition  of  a  dilute  solution  of  sodium 
hydrate,  the  solution  is  colored  yellow  or  yellowish  brown  and  ex- 
hibits a  beautiful  greenish  fluorescence. 

If  the  substance  cannot  be  demonstrated  with  these  tests,  the 
urine  is  acidulated  with  hydrochloric  acid  and  allowed  to  stand 
exposed  to  the  air,  so  that  any  urobilinogen  that  mav  be  present 
is  transformed  into  the  free  pigment.  The  fluid  is  then  examined 
with  the  spectroscope,  when  in  the  presence  of  urobilin  a  distinct 
band  of  absorption  is  obtained  between  b  and  F,  extending  bevond 
F  to  the  right.  A  similar  band  is  also  obtained  in  alkaline  solu- 
tion, but  is  not  so  intense  and  does  not  extend  bevond  F. 

Isolation. — To  isolate  the  pigment,  if  present  in  large  amounts, 
the  urine  is  directly  precipitated  with  ammonia  and  chloride  of 
zinc.  The  precipitate  is  thoroughly  washed  with  water,  extracted 
with  alcohol  bv  boiling,  dried,  and  then  dissolved  in  ammonia.  The 
resulting  solution  ia  precipitated  with  subacetate  of  lead,  the  precipi- 
tate washed  with  water,  and  extracted  with  boiling  alcohol  as  before, 
and  decomposed  with  acid  alcohol.  The  filtered  alcoholic  solution  is 
treated  with  one-half  its  volume  of  chloroform  and  diluted  with 
water;  the  urobilin  passes  into  the  chloroform  on  moderate  agita- 
tion. The  chloroform  solution  is  then  washed  with  water.  On 
in!  distillation  the  pigmenl  remains  as  an  amorphous  reddish  mntc- 
rial,  which  can  be  further  purified  by  washing  with  ether,  which  takes 
up  contaminating  red   pigments.     The  substance  is  readily  soluble 


294  THE   URINE. 

in  alcohol,  amyl  alcohol,  and  chloroform,  less  readily  so  in  water 
and  ether. 

Under  pathological  conditions  still  other  pigments  may  be  found 
in  the  urine.  These  comprise  haemoglobin  and  its  derivatives, 
hsematin,  methaemoglobin,  and  haematoporphyrin  ;  further,  also  uro- 
rubrohaematin  and  urofuscohasmatiu,  which  are  also  undoubtedly 
derived  from  haemoglobin,  but  which  have  thus  far  been  found  in 
the  urine  on  only  one  occasion  ;  further,  the  common  pigments  of  the 
bile ;  and,  finally,  substances  which  belong  to  the  class  of  the 
so-called  melanins.  For  a  consideration  of  the  various  pathological 
conditions  under  which  the  bodies  may  be  met  with,  however,  the 
reader  is  referred  to  special  works  on  diagnosis.  At  this  place  I 
shall  merely  describe  the  more  common  tests  by  which  their 
presence  can  be  demonstrated. 

The  Blood-pigments. — If  the  microscopical  examination  of  the 
urine  shows  the  presence  of  red  blood-corpuscles  in  the  sediment, 
further  chemical  examination  is,  of  course,  unnecessary.  Cases  of 
simple  haemoglobinuria,  in  contradistinction  to  haematuria,  may  occur, 
however,  in  which  dissolution  of  the  haemoglobin  has  taken  place  in 
the  circulation  already,  and  in  which  blood-corpuscles  do  not  appear 
in  the  urine.  In  such  an  event  the  demonstration  of  blood-pig- 
ment can  be  made  only  by  chemical  methods.  Its  presence,  it  is 
true,  is  usually  indicated  by  the  color  of  the  urine,  but  this  may 
be  simulated  by  other  substances  as  well. 

Heller's  Test. — This  is  the  most  convenient  test  for  demon- 
strating the  presence  of  blood-pigment  in  the  urine,  and,  in  the  modi- 
fication here  given,  exceedingly  sensitive.  It  is  based  upon  the 
decomposition  of  the  pigment  in  question  by  means  of  caustic 
alkali  and  the  resulting  formation  of  haemochromogen.  To  this 
end,  a  small  amount  of  the  urine,  or,  still  better,  of  the  sediment,  is 
rendered  strongly  alkaline  with  caustic  alkali  and  boiled.  On  stand- 
ing, the  precipitated  earthy  phosphates  settle  to  the  bottom,  and  are 
colored  a  more  or  less  intense  carmin  by  the  haemochromogen,  which 
has  likewise  separated  out.  That  the  pigment  is  in  reality  haemo- 
chromogen can  be  readily  demonstrated  on  spectroscopic  exami- 
nation (see  page  330).  When  controlled  in  this  manner,  the  test 
is  exceedingly  sensitive,  and  may  still  yield  a  positive  result  even 
when  the  chemical  test  by  itself  does  not  give  a  well-pronounced 
reaction. 

Spectroscopic  Examination. — On  direct  spectroscopic  exam- 
ination the  spectrum  of  methaemoglobin  is  usually  obtained.  The 
urine  should  first  be  acid,  and  if"  necessary  a  little  acetic  acid  is 
added.  On  the  addition  of  a  little  ammonia  and  ammonium  sul- 
phide and  subsequent  filtration  the  broad  band  of  haemoglobin  is  then 
obtained.  With  oxyhaemoglobin,  on  the  other  hand,  the  two  bands 
between  T>  and  E  are  observed ;  and  upon  the  subsequent  addition 
of  ammonia  and  ammonium  sulphide  and  filtration  the  spectrum  of 
reduced  haemoglobin  results.     If  this  does  not  appear  distinctly,  the 


THE  PIGMENTS  OF  THE    URIXE.  295 

solution  is  treated  with   an  excess  of  sodium  hydrate   solution,  and 
will  then  give  the  spectrum  of  haemochromogen. 

Haematin. — Haematin  is  very  rarely  found  in  the  urine.  Its 
presence  as  such  can  be  determined  only  by  spectroscopic  examina- 
tion. Like  haemoglobin  and  methaemoglobin,  it  gives  Heller's 
reaction. 

Haematoporphyrin. — According  to  Garrod,  traces  of  haematopor- 
phvrin  may  be  found  in  every  urine.  Larger  quantities  are 
observed  in  a  number  of  diseases,  but  even  in  these  the  amount 
is  usually  so  small  that  its  presence  will  scarcely  be  suspected 
from  simple  inspection.  Typical  haematoporphyrinuria,  on_  the 
other  hand,  may  be  observed  following  the  prolonged  administra- 
tion of  sulphonal,  trional,  and  tetronal,  or  in  cases  of  poisoning  with 
the  substances  in  question.  The  urine  then  appears  dark  red  in 
color,  and  on  standing  may  turn  almost  black.  As  Hammarsten 
has  pointed  out,  this  change  in  color  is  only  in  part  due  to  haemato- 
porphyrin, and  is  largely  referable  to  other  red  and  reddish-brown 
pigments  of  unknown  character.  Whether  or  not  different  haemato- 
porphyrins  exist  has  not  been  definitely  determined,  but  is  probable. 
In  freshly  voided  urines  haematoporphyrin  probably  exists  in  com- 
bination with  some  other,  still  unknown  body,  with  which  it  forms 
a  colorless  chromogen.  From  this  the  free  pigment  then  develops 
on  exposure  to  the  air. 

Like  the  common  blood-pigments  and  haematin,  haematoporphyrin 
also  reacts  with  Heller's  test.  To  prove  its  presence,  however,  as 
such,  a  spectroscopic  examination  is  necessary.  To  this  end,  the 
urine  is  precipitated  with  barium  hydrate  and  barium  chloride. 
The  precipitate  is  washed  and  allowed  to  stand  in  contact  with 
acidulated  alcohol,  which  extracts  the  pigment.  After  filtering, 
the  solution  is  examined  with  the  spectroscope;  if  subsequently 
the  solution  is  rendered  alkaline  with  ammonia,  the  spectrum  of 
haematoporphyrin  in  alkaline  solution  is  obtained.  To  isolate  the 
substance  as  such,  the  acid  solution  is  mixed  with  a  little  chloro- 
form and  diluted  with  water.  On  gentle  agitation  the  chloroform 
take-;  up  the  greater  portion  of  the  haematoporphyrin,  while  a 
-mill  fraction  and  other  pigments  remain  in  the  diluted  alcoholic 
solution.  On  evaporating  the  chloroform  extract  the  substance  is 
obtained  in  comparatively  pure  form. 

Neumeister  states  that  besides  haematoporphyrin  another  deriva- 
tive of  the  blood-pigment  may  be  observed  in  cases  of  poisoning 
with  sulphonal,  which,  in  contradistinction  to  the  first,  contains  iron. 
This  does  not  read  with  Heller's  lest,  however,  while  the  color 
of  the  urine  is  the  same  as  in  typical  haematoporphyrinuria.  The 
pigment  is  precipitated  by  an  alkaline  barium  chloride  solution, 
and  can    be    subsequently  dissolved    in    acid    alcohol.      This   solution 

presents  ;i  reddish-violel  color,  and  shows  one  broad  band  of  absorp- 
tion in  the  bine  portion  of  the  spectrum  immediately  bordering  on 
the  green. 


296  THE    URINE. 

On  rendering  the  solution  alkaline  with  ammonia  the  pigment  is 
thrown  down.  On  adding  an  excess  of  sodium  hydrate  solution,  on 
the  other  hand,  it  is  dissolved,  while  the  liquid  assumes  a  yellow 
color.  This  solution  then  shows  a  sharp,  narrow  line  in  the  green, 
near  the  blue  portion  of  the  spectrum,  but  disappears  after  the 
solution  has  stood  for  some  time. 

Urorubrohsematin  and  Urofuscohssmatin. — These  pigments  were 
isolated  by  Baumstark  from  the  urine  of  a  leprosy  patient,  but  have 
not  been  encountered  since.  Their  relation  to  hseniatin  is  apparent 
from  the  formula? : 

C32H32]Sr+04Fe,  hsematin  (Nencki  and  Sieber). 

C34H35N405Fe,  bsematin  (Hoppe-Seyler). 

C68PI94X8O30Fe,  urorubrohsematin. 

CG8H106N8O26,  iirofuscohaematin. 

The  pigments  were  isolated  as  follows  :  the  urine,  which  presented 
a  color  varying  from  a  dark  red  to  a  brownish  red,  was  dialyzed  and 
the  final  contents  of  the  dialyzer  dissolved  in  sodium  hydrate  solu- 
tion ;  upon  the  addition  of  hydrochloric  acid  to  this  solution 
urofuscoheematin  separated  out  in  brown  flakes,  while  the  second 
pigment  remained  in  solution,  coloring  this  a  beautiful  red.  After 
filtering  off  the  first,  the  solution  was  again  dialyzed,  when  the 
second  pigment  separated  out. 

Whether  or  not  any  relation  exists  between  these  two  bodies  and 
hsematoporphyrin  in  impure  form,  as  Hammarsten  suggests,  must 
remain  an  open  question. 

Melanins. — Notably  in  association  with  the  existence  of  melanotic 
tumors,  but  at  times  also  in  other  diseases  which  are  associated  with 
an  increased  destruction  of  red  blood-corpuscles,  urines  are  met  with 
which  gradually  turn  a  dark  brown  or  black  on  standing.  When 
freshly  voided,  however,  they  commonly  present  a  normal  color. 
The  pigment  or  pigments  which  are  thus  formed  belong  to  the  class 
of  melanins,  and  are  identical  with  those  which  can  be  obtained 
from  the  pigmented  growths.  They  are  probably  eliminated  in 
combination  with  some  other  substance  which  is  as  yet  unknown,  as 
colorless  melanoc/ens  and  from  which  the  free  pigments  are  obtained 
on  oxidation.  They  are  unquestionably  derived  from  the  common 
pigments  of  the  blood,  but  are  individually  little  known. 

To  prove  that  the  change  in  the  color  of  the  urine  is  referable  to 
melanins,  a  fresh  specimen  should  be  procured,  and  treated  with 
bromine-water.  If  the  chromogens  in  question  are  present,  the 
resulting  precipitate,  which  is  yellow  at  first,  turns  black  on  standing. 
On  the  addition  of  a  few  drops  of  a  strong  solution  of  ferric 
chloride  a  similar  reaction  is  obtained. 

To  isolate  the  pigments  from  the  urine,  the  fluid  is  first  precipi- 
tated with  an  alkaline  solution  of  barium  chloride.  From  the 
resulting  precipitate  the  pigments  are  extracted  with  a  concentrated 


THE  PIGMENTS   OF  THE    URINE.  297 

solution  of  sodium  carbonate,  and  are  then  precipitated  by  adding 
an  excess  of  sulphuric  acid.  By  redissolution  in  a  dilute  solution 
of  sodium  hydrate  and  reprecipitation  with  acetic  acid  they  can  be 
obtained  in  comparatively  pure  form.  But  it  "will  be  noted  that  a 
certain  fraction  remains  in  the  acetic  acid  solution,  which  indicates 
the  existence  of  at  least  two  different  pigments.  The  soluble 
form  has  been  termed  phymatorhusin,  and,  according  to  Nencki 
and  Sieber.  contains  no  iron,  while  Morner  claims  that  this  is 
present.  Elementary  analysis  of  this  pigment  has  given  the  fol- 
lowing results  (Morner) : 

From  growth.  From  urine. 

Carbon       55.32  to  56.13  per  cent.  55.76  per  cent. 

Hvdrogen      5.65  to    6.33   "      "  5.95  "       " 

Nitrogen 12.30  "      "  12.27   "        " 

Sulphur 7.97  "      "  9.01   "        " 

Iron 0.063  to  0.081  "      "  0.20  " 

From  melanotic  growths  in  horses  a  hippomelanin  has  been 
obtained,  which,  in  contradistinction  to  the  first,  is  soluble  in  solu- 
tions of  the  alkalies  with  difficulty. 

The  Bile-pigments. — Bile-pigments  are  never  found  in  the 
urine  under  normal  conditions.  As  a  rule,  freshly  voided  urine 
contains  only  bilirubin.  If  a  complicating  cystitis,  however,  exists, 
the  common  derivatives  of  bilirubin,  viz.,  biliverdin,  bilifuscin, 
biliprasin,  and  bilihumin,  may  also  be  encountered. 

Bile-containing  urines  present  a  very  characteristic  color,  which 
may  vary  from  a  bright  golden-yellow  to  a  greenish  brown,  and  on 
microscopical  examination  it  is  common  to  find  the  morphological 
elements  stained  an  intense  yellow.  This  color  is  further  imparted 
to  the  foam  on  shaking.  But  as  urobilin  when  present  in  large 
amounts  may  impart  a  similar  color  to  the  urine,  it  is  always 
better  to  resort  to  chemical  tests.  These  have  been  described  in 
detail  in  the  section  on  the  Bile,  and  are  directly  applicable  also  to 
the  urine  (see  page  158). 

Oilier  pigments  also  may  occur  in  the  urine  after  the  ingestion  of 
various  drugs,  but  as  the  products  thus  formed  are  of  no  special 
interesl  from  the  standpoint  of  animal  chemistry,  they  are  not  con- 
sidered  at  this  place. 

The  Bile-acids. — The  occurrence  of  bile-acids  in  the  urine  is 
solely  a  pathological  phenomenon.  In  normal  urines  they  arc  never 
found,  and  even  in  complete  obstruction  of  the  common  duct  their 
amount  i-  quite  .-mall.  To  demonstrate  their  presence,  they  must 
firsl  be  isolated  as  Platner'a  bile,  and  can  then  he  identified  by 
polarimetric  examination,  their  action  upon  the  frog's  heart,  etc. 
[see  pages   117  and   148). 


298  THE   URINE. 

Fats,  Cholesterin,  and  Lecithins. 

Fats. — Traces  of  fat  may  be  observed  in  the  urine  under  normal 
conditions  when  excessive  amounts  have  been  ingested.  During 
pregnancy  also  a  lipuria  has  been  noted,  and  is  probably  associated 
with  the  development  of  the  function  of  lactation.  Otherwise  the 
condition  is  essentially  a  pathological  phenomenon,  and  is  notably 
observed  in  acute  yellow  atrophy,  in  phosphorus  poisoning,  follow- 
ing fracture  of  the  long  bones,  etc.  The  largest  amounts,  however,, 
are  found  in  cases  of  so-called  chyluria.  Owing  to  the  existence  of 
the  fats  in  fine  emulsions,  such  urines  may  resemble  milk  in  their 
general  appearance,  and  on  standing  a  layer  of  "cream"  forms  at 
the  top. 

To  establish  the  presence  of  fats,  the  surface  layer  of  the  urine  is 
extracted  with  ether,  the  ether  is  evaporated,  and  the  residue  brought 
in  contact  with  a  piece  of  paper,  when  characteristic  stains  result. 

Cholesterin. — Cholesterin  is  very  rarely  found  in  the  urine,  and 
has  thus  far  been  encountered  only  under  pathological  conditions. 
It  probably  always  occurs  in  crystalline  form,  and  is  thus  readily 
recognized.  If  any  doubt  exists,  the  substance  in  question  is 
examined  as  has  been  described  (page  163). 

Lecithins. — Lecithins  as  such  have  been  observed  in  the  urine 
only  in  chyluria,  where  they  are  commonly  present  in  association 
with  cholesterin  and  fat.  One  of  the  derivatives  of  lecithin,  how- 
ever, is  found  also  in  the  urine  under  normal  conditions,  though 
in  very  small  amounts.  This  is  glycerin-phosphoric  acid.  It  is  no 
doubt-  referable  to  decomposition  of  the  lecithins  of  the  food  in  the 
intestinal  canal,  but  may  at  times  also  be  derived  from  the  lecithins 
of  the  tissues.  To  demonstrate  its  presence,  several  liters  of  urine 
are  freed  from  the  common  phosphates  by  rendering  the  urine 
alkaline  with  barium  hydrate  and  precipitating  the  heated  mixture 
with  barium  chloride.  The  excess  of  barium  is  removed  with 
carbonic  acid  and  the  filtrate  evaporated  to  a  syrup.  This  is  ex- 
tracted with  absolute  alcohol,  when  the  remaining  material  is  dis- 
solved in  a  little  water  and  boiled  with  hydrochloric  acid.  The 
glycerin-phosphoric  acid  is  thus  decomposed,  with  the  liberation  of 
glycerin  and  phosphoric  acid.  On  evaporating  to  dryness,  the 
residue  is  extracted  with  water,  and  the  presence  of  phosphoric 
acid  demonstrated  in  the  aqueous  solution  by  the  usual  tests. 

Ferments. 

Every  urine  contains  ferments  which  are  thought  to  be  identical 
with  the  pepsin,  ptyalin,  and  chymosin  of  the  digestive  fluids.  It 
can  be  shoAvn,  as  a  matter  of  fact,  that  substances  are  present 
which  are  capable  of  digesting  fibrin  in  acid  solution,  of  inverting 
starch  to  maltose,  and  of  coagulating  milk.  There  is  no  proof, 
however,  that  these  bodies  are  derived  from  the  digestive  glands, 
as  Neumeister  and  others  claim. 


THE  PTOMAINS   OF  THE   URINE.  299 

Gases. 

In  normal  urine  a  certain  amount  of  oxygen,  nitrogen,  and  nota- 
bly of  carbon  dioxide  is  found  in  solution,  and  can  be  withdrawn 
by  the  air-pump.  Under  pathological  conditions,  further,  a  variable 
amount  of  hydrogen  sulphide  may  be  encountered.  This  notably 
occurs  in  cases  of  cystitis,  in  which  the  decomposition  of  albumin 
and  sulphur  bodies  may  already  take  place  within  the  bladder,  owing 
to  the  activity  of  various  micro-organisms.  But  in  a  few  isolated 
cases  the  gas  was  apparently  derived  from  the  intestinal  tract,  and 
absorbed  either  directly  from  the  rectum  or  indirectly  from  the 
blood. 

All  urines  when  exposed  to  the  air  sooner  or  later  contain  hydro- 
gen sulphide  in  the  free  state,  which  is  referable,  as  stated  above, 
to  the  action  of  certain  micro-organisms.  Especially  large  amounts 
are  observed  when  cystin-containing  urines  are  thus  allowed  to 
undergo  decomposition.  To  test  for  hydrogen  sulphide,  a  strip  of 
filter-paper  is  moistened  with  a  few  drops  of  a  solution  of  sodium 
hydrate  and  one  of  lead  acetate,  and  is  then  clamped  in  the 
neck  of  the  bottle  containing  the  urine.  If  the  gas  in  question  is 
present,  the  paper  is  colored  a  grayish  brown  or  black,  owing  to 
the  formation  of  lead  sulphide.  When  present  in  large  amounts 
it  is  detected  also  by  its  odor. 

Ptomains. 

So  far  as  known,  ptomains  are  not  found  in  the  urine  under 
normal  conditions.  In  disease,  however,  various  basic  substances 
have  been  encountered  which  supposedly  belong  to  this  class.  But 
with  the  exception  of  cadaverin  and  putrescin,  which,  as  has  been 
stilted,  may  occur  in  association  with  cystinuria,  these  bodies  have 
l)icii  isolated  in  amounts  scarcely  sufficient  to  establish  their 
chemical  nature.  This  holds  good  more  especially  of  the  bodies 
which  Griffith  claims  to  have  isolated  from  the  urine  of  patients 
suffering  from  scarlatina,  measles,  mumps,  carcinoma,  etc. 

A-  regards  the  origin  of  putrescin  and  cadaverin  in  cystinuria,  the 
opinion  prevails  that  they  are  due  to  a  specific  form  of  intestinal 
putrefaction.  This  is,  however,  not  necessarily  the  case,  and  in  my 
opinion  the  diaminuria,  like  the  cystinuria,  is  the  expression  of  a 
distincl  metabolic  disturbance.  I  have  pointed  out  that  both  diamins 
can  l»e  derived  from  arginin  and  lysin  in  the  laboratory,  and  there 
is  every  reason  to  suppose  that  the  same  transformation  can  also 
occur  in  the  living  organism.  That  arginin  at  least  actually  occurs 
in  the  tissues  of  the  body  has  been  demonstrated  by  Gulewitch,  who 

found  the  substance  in   the  spleen. 

The-  quantity  of  the  diamins  which  may  be  eliminated  in  the 
mine  in  cases  of  cystinuria  is  quite  variable.  On  some  days  traces 
only  or  none  ;it  nil  i-  found,  while  at  other  times  very  considerable 
amount-  may  be  obtained.     In  one  of  mycases  I  was  able  to  isolate 


300  THE   URINE. 

1.6  grammes  of  the  benzoylated  cadaverin  from  the  collected  urine 
of  twenty-four  hours. 

To  demonstrate  the  presence  of  diamins,  the  method  of  Bau- 
mann  and  v.  Udranszky  is  most  conveniently  employed.  To  this 
end,  the  collected  urine  of  twenty-four  hours  or  more  is  benzoylated 
by  shaking  with  benzoyl  chloride  in  the  presence  of  sodium  hydrate. 
As  a  general  rule,  25  c.c.  of  the  chloride  and  200  c.c.  of  a  10  per 
cent,  solution  of  sodium  hydrate  are  used  for  1500  c.c.  of  the  urine. 
The  resulting  precipitate,  which  contains  the  earthy  phosphates,  the 
benzoylated  carbohydrates  which  are  normally  present  in  every 
urine,  and  the  greater  portion  of  the  benzoylated  diamins,  is  then 
filtered  oiF,  extracted  with  boiling  alcohol,  filtered,  and  the  alcoholic 
extract  concentrated  on  a  water-bath.  This  solution  is  then  poured 
into  thirty  times  its  volume  of  water.  On  standing,  the  benzoy- 
lated diamins  separate  out  in  crystalline  form,  and  are  then  freed 
from  adhering  carbohydrates  by  repeated  solution  in  alcohol  and 
precipitation  in  water.  The  process  is  continued  until  the  desired 
degree  of  purity  is  obtained.  The  resulting  crystals  are  finally 
filtered  off,  dried  over  sulphuric  acid,  and  identified  by  their 
melting-point  and  the  contained  amount  of  nitrogen.  If  both 
diamins  are  present,  the  crystals  lose  their  water  of  crystalliza- 
tion at  120°  C,  and  melt  at  140°  C.  To  separate  them  from 
each  other,  they  are  dissolved  in  a  little  warm  alcohol,  and  are 
treated  with  twenty  times  as  much  ether.  Benzoyl  putrescin  is 
thus  thrown  down,  while  the  cadaverin  compound  remains  in  solu- 
tion. The  crystals  of  the  former  melt  at  175°-176°  C,  while  the 
melting-point  of  the  latter  lies  between  129°  and  130°  C. 

A  small  portion  of  the  diamins  remains  in  the  first  filtrate.  To 
isolate  these,  the  liquid  is  acidified  with  sulphuric  acid  and  ex- 
tracted with  ether.  The  ethereal  extract  is  evaporated,  and  the 
final  solution,  before  congealing,  placed  in  as  much  of  a  12  per 
cent,  solution  of  sodium  hydrate  as  is  required  for  its  neutralization. 
From  three  to  four  times  as  much  of  the  alkali  solution  is  then 
added.  On  standing  in  the  cold,  sodium  benzoyl-cystin  separates 
out,  together  with  the  benzoylated  diamins.  The  crystals  are  fil- 
tered off  and  placed  in  cold  water.  This  dissolves  the  cystin  com- 
pound, while  the  diamins  remain  undissolved.  They  are  soluble  in 
warm  alcohol,  and  can  then  be  separated  from  each  other,  as  has 
been  described. 


CHAPTER    XIII. 

THE  ANIMAL  CELL. 

The  cell  constitutes  the  morphological  unit  of  all  animal  and 
vegetable  life,  and  as  such  is  capable  of  manifesting  those  peculiar 
activities  which  we  regard  as  characteristic  of  living  matter.  In 
its  simplest  form  it  represents  a  tiny  bit  of  a  more  or  less  granular, 
gelatinous  substance — the  so-called  protoplasm — in  the  interior  of 
which  a  somewhat  more  solid-looking  body  can  be  made  out,  which 
is  termed  the  nucleus.  Such  simple  cells  exist  in  nature,  either  as 
such  or  as  conglomerations  of  many  cells  which  represent  the  higher 
forms  of  animal  and  vegetable  life.  All  living  matter,  however, 
whether  simple  or  complex,  lias  for  its  origin  the  single  cell.  But 
while  in  the  lowest  forms  of  life  the  single  cell  is  capable  of  per- 
forming all  those  functions  which  are  characteristic  of  living  matter 
by  itself,  we  find,  as  we  ascend  in  the  scale  of  animal  and  vegetable 
life,  that  certain  groups  of  cells  are  here  set  aside  for  the  purpose 
of  executing  separate  functions.  Such  groups  of  cells  we  speak  of 
a-5  tissues,  and  we  accordingly  find  in  the  highly  organized  mammal 
a  differentiation  of  the  entire  body  into  tissues,  which  according 
to  their  functions  may  be  grouped  as  tissues  of  locomotion,  of  re- 
production, of  digestion,  of  excretion,  etc.  With  such  a  differentia- 
tion of  cells  into  tissues,  however,  the  original  aspect  of  the  cell  is 
m  >re  or  less  changed.  The  highly  differentiated  voluntary  muscle- 
cell  would  thus  at  first  sight  scarcely  be  recognized  as  being  in  any 
way  related  to  the  oval  cell  from  which  it  is  primarily  derived. 
On  careful  examination,  however,  we  find  that,  no  matter  how 
unlike  its  ancestral  cell  such  a  specialized  cell  may  appear,  the  dif- 
ference is  only  apparent,  for  all  cells  of  the  body  consist  at  one 
time  of  i heir  existence  at  least  of  protoplasm  and  nucleus.  The 
striated  portion  of  the  muscle-cell  is  thus  nothing  more  than  the 
protoplasm  of  the  original  cell,  differentiated  and  modified  in  accord- 
ance with  the  function  which  the  cell  is  to  perform.  In  some  cells, 
however,  such  as  those  of  the  adipose  tissue,  the  original  differentia- 
tion into  protoplasm  and  nucleus  is  apparently  lost,  and  on  ordinary 
microscopical  examination  it  appears  that  such  cells  are  nothing  but 
large  globules  of  fat.  Bui  with  special  methods  of  staining  we  can 
demonstrate  even  here  that  there  area  nucleus  and  protoplasm.    The 

only  cells,  in  fact,  in  which  a  nucleus  cannot  always  be  demonstrated 
are  tin-  red  corpuscles  of  the  circulating  blood  of  man  and  the 
anthropoid    ape-.      We   find,    however,    that    even    in   adult    man   all 

301 


302  THE  ANIMAL   CELL. 

red  corpuscles  at  one  period  of  their  existence,  viz.,  in  their  juve- 
nile form,  are  nucleated,  and  that  under  certain  conditions,  as  after 
copious  hemorrhages,  such  nucleated  corpuscles  may  occur  in  the 
circulating  blood  in  large  numbers.  In  the  bone-marrow,  where 
they  are  apparently  formed,  they  are  always  present. 

As  all  manifestations  of  life  are  intimately  associated  with 
chemical  changes  which  bring  about  a  transformation  of  potential 
into  kinetic  energy,  such  changes  must  of  necessity  occur  in  every 
living  cell.  These  changes,  moreover,  must  vary  with  the  func- 
tion which  the  cell  is  to  perform,  and  will  hence  differ,  to  a 
certain  extent  at  least,  with  the  different  tissues  of  the  complex 
organism.  In  the  monocellular  organisms,  where  all  the  various 
functions  are  performed  by  the  single  cell,  all  these  varying  changes 
must  hence  be  represented.  But  it  is  to  be  inferred  that  in  accord- 
ance with  the  greater  simplicity  in  structure  the  chemical  changes 
also  must  be  of  a  simpler  character.  We  should  hence  expect  that  a 
study  of  the  chemical  processes  which  take  place  in  such  low  forms 
of  life  would  furnish  us  with  a  better  insight  into  the  metabolism 
of  the  complex  organism  than  could  be  attained  from  an  investiga- 
tion of  the  higher  forms.  Unfortunately,  however,  this  is  almost 
an  impossibility  with  the  usual  chemical  and  physical  methods,  for 
in  attempting  such  a  study  we  are  met  with  a  most  serious  obstacle, 
viz.,  our  inability  to  maintain  the  life  of  the  individual  cell  during 
such  an  investigation.  We  would  consequently  have  no  proof  that 
those  products  which  we  could  isolate  from  the  dead  cells  were 
present  as  such  in  the  living  organism.  The  technical  difficulties, 
moreover,  which  stand  in  the  way  of  such  a  study  are  almost  insur- 
mountable. With  microchemical  methods,  it  is  true,  something 
more  definite  might  be  accomplished,  and  although  this  branch  of 
investigation  is  still  in  its  infancy,  it  has  already  furnished  us  with 
a  certain  amount  of  valuable  information.  The  great  advances 
which  have  thus  been  made  in  our  knowledge  of  the  structure 
of  cells  have  largely  been  accomplished  in  this  manner.  Upon 
the  chemical  processes  themselves,  however,  which  take  place 
in  the  cell,  not  much  light  has  as  yet  been  thrown  in  this 
manner.  We  are  consequently  dependent  for  our  knowledge  of 
the  metabolic  processes  which  take  place  in  the  living  body  upon 
a  study  of  the  individual  tissues  as  such,  and  the  changes  which 
result  in  certain  substances  when  introduced  from  without. "  An 
analysis  of  these  tells  us  in  what  form  the  various  food-stuffs  are 
represented  in  the  individual  cells.  By  then  studying  the  vari- 
ous decomposition-products  which  can  be  isolated  from  the  tissues, 
we  can  in  a  measure  form  an  idea  of  the  manner  in  which  these 
products  were  produced  and  of  the  form  in  which  they  were 
represented  in  the  original  and  more  complex  molecule.  With 
some  tissues,  however,  this  is  more  difficult  than  with  others.  The 
most  satisfactory  results,  on  the  whole,  regarding  the  chemical 
structure  of  the  individual  cell  have  thus  far  been  obtained  from 


THE  ANIMAL   CELL.  303 

an  investigation  of  those  tissues  which  are  especially  rich  in  cells, 
and  in  which  the  cells  can  he  more  or  less  completely  separated  from 
the  underlying  matrix  and  from  other  components  which  may  be 
present  at  the  same  time.  This  is  especially  true  of  the  leucocytes 
of  the  blood.  As  these  bodies,  moreover,  are  but  little  differentiated, 
they  may  well  serve  as  types  of  primitive  cells.  They  are  all 
nucleated,  and  contain  a  varying  amount  of  protoplasm,  which  in 
some  is  capable  of  progressive  movement.  A  limiting  membrane, 
as  in  most  animal  cells,  does  not  exist.  But  it  is  generally  supposed 
that  a  meshwork  of  fine  fibrils  pervades  the  protoplasm,  and  that  in 
the  meshes  a  more  liquid  portion  is  contained.  This  is  termed  the 
hyaloplasm,  in  contradistinction  to  the  more  solid  spongioplasm.  In 
some  forms  the  protoplasm  is  apparently  perfectly  homogeneous,  while 
in  others  it  is  studded  with  numerous  granules  of  variable  size,  which 
execute  more  or  less  active  oscillatory  movements,  which  are  spoken 
of  as  the  molecular  movements  of  Brown. 

The  reaction  of  the  protoplasm  is  alkaline,  while  the  nucleus 
apparently  contains  no  free  alkali.  This  may  be  shown  by  staining 
dried  blood  films  with  a  solution  of  acid  erythrosin  in  chloroform, 
when  it  will  be  seen  that  the  body  of  the  cell  is  colored  a  bright  red, 
while  the  nucleus  is  not  stained.  The  most  intense  reaction  is 
obtained  with  the  protoplasm  of  the  so-called  lymphocytes. 

The  granules  which  are  found  in  certain  forms  of  leucocytes  are 
apparently  of  an  albuminous  nature.  According  to  their  affinity  for 
acid,  basic,  or  neutral  dyes,  they  are  termed  oxyphilic,  basophilic, 
ami  neutrophilic,  respectively.  Fatty,  mineral,  or  pigment  granules, 
which  may  be  found  in  other  animal  cells,  are  usually  not  seen  in 
leucocytes.  In  the  eosinophilic  leucocytes,  however,  the  presence  of 
iron  can  readily  be  demonstrated  by  microchemical  methods.  In 
another  form  it  seems  to  be  present  in  all  varieties  of  cells,  and  is 
especially  abundant  in  the  nuclei. 

In  the  mineral  ash  we  further  find  potassium,  sodium,  calcium, 
magnesium,  phosphorus,  and  chlorine,  and  it  is  to  be  noted  that,  in 
contradistinction  to  the  animal  fluids,  the  cell  contains  a  relatively 
larger  amount  of  potassium  and  phosphorus,  while  sodium  and 
chlorine  are  more  abundant  in  the  fluids.  That  the  phosphates  are 
of  prime  importance  in  the  life  of  the  cell  is  now  definitely 
established,  and  Loew  showed  that  in  the  spirogyra,  for  example, 
growth  and  cellular  division  are  greatly  interfered  with  by  their 
absence.  The  importance  of  the  phosphates  is  without  doubt  con- 
nected  with  the   presence  of  the  nucleins  in  the  nuclei — i.  e.,  of 

albuminous    substances   which,  as  we    have   seen,  contain  a  relatively 

large  amount  of  phosphorus  in  organic  combination. 

The  protoplasm  of  the  cell  is  very  rich  in  water,  and,  in    addition 

to  small  an nt-   of  mineral    -alt;-,  consists    essentially  of  albumins. 

Some  of  these  are  albumins  proper,  but  the  greater  portion  by 
far  i-  represented  1a-  substances  which  belong  to  the  proteid 
group.      It  appears,  moreover,  that  the  traces  of  serum-albumin  and 


304  THE  ANIMAL  CELL. 

globulin  which  are  present  do  not  represent  integral  constituents  of 
the  living  protoplasm,  but  are  merely  to  be  regarded  as  food-stuffs, 
or  possibly  even  as  decomposition-products  of  the  protoplasmic 
molecule. 

The  proteids  which  are  here  found  principally  belong  to  the 
nucleo-albumins,  and  it  is  to  be  noted  that,  in  contradistinction  to 
those  which  occur  in  the  nucleus,  these  nucleo-albumins  contain 
relatively  but  little  phosphorus.  The  albuminous  radicle  in  one  of 
them  at  least  is  quite  constantly  a  vitellin.  Gluco proteids  may  also 
be  present,  but  are  not  so  constant  as  the  nucleo-albumins. 

Of  other  constituents  of  the  protoplasm,  lecithin  is  the  most 
constant.  In  addition  we  find  certain  protagons,  glycogen,  choles- 
terins,  and  in  dead  cells  also  paralactic  acid,  to  which  the  acid 
reaction  of  dead  protoplasm  is  due. 

The  total  amount  of  solids,  including  the  mineral  salts,  which  are 
thus  found  in  protoplasm,  is  always  small,  and  probably  never 
exceeds  15—20  per  cent.,  while  the  remaining  weight  is  referable  to 
water.  In  some  instances  indeed  almost  the  entire  cell  is  taken  up 
by  the  nucleus,  and  in  the  lymphocytes,  for  example,  only  1.76 
per  cent,  of  the  total  79.21  per  cent,  of  albumins,  as  calculated  for 
the  dry  material,  is  present  in  the  protoplasm. 

The  nucleus  may  be  regarded  as  the  essential  living  part  of  all 
animal  and  vegetable  cells,  and  from  it,  no  doubt,  the  various  func- 
tions of  the  cell  as  a  whole  are  directed.  It  is  intimately  connected 
with  the-process  of  reproduction,  and  during  this  process  it  under- 
goes a  series  of  most  remarkable  changes,  which  are  collectively 
termed  the  karyokinesis  or  karyomitosis  of  the  nucleus.  Micro- 
scopically the  quiescent  nucleus  represents  a  round  or  oval  little 
body,  which  usually  occupies  an  excentric  position  within  the  cell. 
It  is  surrounded  by  a  nuclear  membrane,  and  contains  a  meshwork 
of  extremely  fine  fibrils,  and  one  or  more  nucleoli.  Both  fibrils  and 
nucleoli  possess  a  marked  affinity  for  anilin  dyes,  while  the  nuclear 
membrane  and  the  more  liquid  hyaloplasm  within  the  nuclear 
meshes  are  scarcely  stained  at  all.  We  therefore  recognize  in  the 
nucleus  the  existence  of  chromatic  and  achromatic  substances,  which 
are  usually  spoken  of  as  the  nuclear  chromatins  and  achromatins. 
During  the  process  of  division  a  peculiar  spinclle-like  body  is  also 
observed  in  the  nucleus,  which,  like  the  nuclear  hyaloplasm,  is 
achromatic. 

In  contradistinction  to  the  cellular  protoplasm, 'the  nucleus  con- 
tains a  much  larger  quantity  of  solids,  but  here  as  there  the  albu- 
mins stand  in  the  foreground.  Whether  or  not  native  albumins 
also  occur  in  the  nucleus  is  not  definitely  known,  but  it  is 
generally  assumed  that  this  is  not  the  case.  The  proteids,  on  the 
other  hand,  are  abundant  and  largely  represented  by  the  nucleins 
and  the  nucleo-albumins.  The  nucleins  indeed  are  thought  to  con- 
stitute the  greater  portion  of  the  chromatic  constituents  of  the 
nucleus,  and  among  them  the  so-called  plastin  apparently  occupies 


THE  ANIMAL   CELL.  305 

a  prominent  position.  This  substance,  while  not  definitely  known, 
is  usually  classed  as  a  nuelein,  but  differs  from  the  more  common 
forms  in  being  soluble  with  difficulty.  Especially  abundant  also  is 
a  nucleo-alburnin,  which  Kossel  and  Lilienfeld  first  obtained  from 
the  thymus  gland  of  the  calf.  This  they  termed  nucleohiston,  from 
the  fact  that  on  treatment  with  hydrochloric  acid  it  is  decomposed 
into  a  nuelein — leukonuclein,  and  a  special  albumose-like  substance — 
biston,  which  differs  from  other  albumins  in  being  insoluble  in  am- 
monium hydrate  (see  page  322).  This  substance  is  probably  iden- 
tical with  "the  so-called  tissue  fibrinogen  and  cellular  fibrinogen  of 
other  observers,  and,  no  doubt,  is  closely  related  to  the  cytoglobin 
and  prseglobulin  of  Alexander  Schmidt. 

In  addition  to  these  substances,  we  further  meet  with  nucleinic 
acids  in  the  free  state,  and  in  some  cells  also  with  the  basic  radicles 
of  the  nucleinic  acids — i.  e.,  the  xanthin  bases,  as  such. 

Whether  lecithins,  protagon,  and  glycogen,  which  are  constantly 
found  in  the  cellular  protoplasm,  likewise  occur  in  the  nucleus,  is 
not  known. 

Of  mineral  constituents,  iron  is  constantly  present,  and  appar- 
ently occurs  in  combination  with  the  nucleins  in  organic  form. 


CHAPTER    XIV. 

THE   BLOOD. 

General  Considerations. — The  blood  of  man  and  of  almost  all 
vertebrate  animals  is  a  slightly  viscid,  somewhat  opaque-looking 
fluid,  which,  according  to  its  origin  from  an  artery  or  a  vein,  pre- 
sents a  color  that  varies  from  a  bright  scarlet  to  a  dark  bluish-red. 
On  microscopical  examination  it  is  seen  to  contain  a  large  number 
of  cellular  elements,  which  are  partly  colored  and  partly  colorless. 
The  former,  which  greatly  predominate,  are  the  red  corpuscles,  or 
erythrocytes  of  the  blood.  In  man  they  are  homogeneous,  normally 
non-nucleated,  circular,  biconcave  disks,  measuring  on  an  average 
7.5  p.  in  diameter.  When  viewed  through  the  microscope  they  are 
of  a  faint  greenish-yellow  color,  while  en  masse  they  present  the 
ordinary  color  of  the  blood.  The  colorless  bodies,  or  leucocytes,  on 
the  other  hand,  are  all  nucleated  and  in  part  capable  of  executing 
amoeboid  movements.  Some  of  them  are  of  about  the  same  size 
as  the  red  corpuscles,  while  others  are  larger.  The  nucleus  may 
be  single  or  multiple,  and  it  will  be  noted  that  in  the  mononuclear 
forms  the  surrounding  protoplasm  is  more  or  less  homogeneous, 
while  in  the  polynuclear  varieties  it  is  distinctly  granular.  The 
total  number  of  the  leucocytes  per  cubic  millimeter  varies  under 
normal  conditions  between  3000  and  10,000,  and  is  thus  much 
smaller  than  the  number  of  the  red  corpuscles,  which  is  generally 
placed  at  between  4,000,000  and  5,000,000  in  the  same  volume  of 
blood. 

In  addition  to  the  red  corpuscles  and  the  leucocytes,  we  further 
find  a  large  number  of  minute  colorless  disks,  measuring  less  than 
one-half  the  diameter  of  a  red  corpuscle,  and  usually  occurring  in 
bunches  of  from  six  to  a  dozen  or  more.  They  are  termed  the 
plaques  or  blood-plates  of  Bizzozero.  On  an  average,  about  635,000 
are  found  in  the  cbmm.  Other  morphological  elements  are  not 
found  in  the  blood  under  normal  conditions,  while  in  disease  nucleated 
red  corpuscles,  both  of  the  adult  and  the  embryonic  type,  as  well  as 
other  forms  of  leucocytes,  may  be  encountered. 

When  blood  is  drawn  from  the  living  body  and  is  allowed  to 
stand,  it  will  be  noted  that  after  a  variable  length  of  time  the  entire 
mass  is  transformed  into  a  semisolid,  jelly-like  material,  wrhich  is 
termed  the  -placenta  sanguinis  or  blood-clot.  On  microscopical  ex- 
amination this  will  be  seen  to  consist  of  a  dense  network  of  fibres, 
in  the  meshes  of  which  the  corpuscles  of  the  blood  are  found.  If 
the  clot  is  now  carefully  separated  from  the  walls  of  the  vessel,  it 

306 


GENERAL  CONSIDERATIONS.  307 

undergoes  shrinkage,  and  presses  out  from  its  meshes  a  clear,  straw- 
colored  fluid,  which  is  termed  the  blood-serum.  This  gradually 
increases  in  amount,  while  the  size  of  the  clot  diminishes,  and  may 
then  be  utilized  for  the  purpose  of  chemical  examination.  The 
fibrous  material  which  is  formed  during  the  process  of  clotting  is 
termed  fibrin.  Its  formation  is  intimately  associated  with  the  death 
of  the  organism,  either  local  or  general,  and  is  dependent  in  the  first 
instance  upon  the  presence  of  a  specific  ferment — the  so-called  fibrin 
ferment,  or  thrombin  of  Alexander  Schmidt.  In  the  circulating 
blood  fibrin  is  not  found,  but  we  here  meet  with  its  mother-substance, 
fibrinogen,  which  is  not  present  in  the  blood-serum.  The  blood- 
plasma,  viz.,  the  fluid,  non-cellular  portion  of  the  circulating  blood, 
thus  differs  from  the  blood-serum  in  containing  fibrinogenic  material, 
but  not  the  fibrin  ferment,  which  is  found  in  the  serum.  The  fer- 
ment itself  results  from  decomposition  of  the  cellular  elements  of 
the  blood,  notably  of  the  blood-plates.  This  may  be  seen  when 
the  process  of  coagulation  is  observed  through  a  microscope.  After 
a  variable  length  of  time,  more  rapidly  when  the  blood-drop  is  not 
too  small  and  when  the  surface  of  the  glass  is  a  little  uneven,  fine 
filaments  of  fibrin  thus  begin  to  appear,  which  usually  have  for  their 
starting-point  those  bunch-like  conglomerations  of  the  plaques  which 
have  already  been  described.  In  these  bodies  the  pro-enzyme  of  the 
ferment,  the  so-called  prothrombin  of  Alexander  Schmidt,  is  proba- 
bly contained,  and  gives  rise  to  the  ferment  itself  when  the  death 
of  the  cell  occurs.  It  should  be  stated,  however,  that  the  fibrin  fer- 
ment is  not  only  derived  from  the  plaques,  but  may  also  be  formed 
during  decomposition  of  the  remaining  cellular  elements  of  the 
blood,  and,  to  judge  from  recent  observations,  from  protoplasmic 
material  in  general  (see  page  325). 

PHYSICAL  CHARACTERISTICS  OF  THE  BLOOD. 

Color. — The  color  of  normal  blood  is  referable  to  the  presence 
of  a  peculiar  albuminous  substance  in  the  red  corpuscles  belong- 
ing to  tin.'  class  of  proteids  which  is  termed  haemoglobin.  In 
arterial  Mood  this  is  principally  found  in  combination  with  oxy- 
gen as  oxyhemoglobin,  while  in  venous  blood  a  mixture  of  both 
occurs.  With  a  preponderance  of  oxyhemoglobin  over  hemoglobin 
the  color  of  the  blood  tends  toward  a  bright  scarlet-red.  In  its 
>nce  ii  assumes  a  dark-bluish  color,  and  we  accordingly  find  all 
gradationa  in  shade  between  the  two.  When  venous  blond  is 
exposed  to  the  air  the  hemoglobin  immediately  absorbs  oxygen, 
and  ie  transformed  into  oxyhemoglobin.  This  actually  takes  place 
in  the  alveoli  of  the  lungs,  and  explain-  the  difference  in  color 
between  the  blood  of  ih<-  right  and  the  l<f't  heart. 

Under  pathological  conditions  we  may  find  -till  other  colors  than 
those  which  have  been  described.  In  coal-gas  poisoning  the  blood 
is  thus  of  a  bright  cherry-red;  in  poisoning  with  potassium  chlorate, 


308  THE  BLOOD. 

anilin,  hydrocyanic  acid,  nitrobenzol,  etc.,  it  is  of  a  brownish-red  or 
a  chocolate  color.  These  changes  are,  as  we  shall  presently  see,  due 
to  certain  chemical  compounds  of  haemoglobin  which  are  not  nor- 
mally found  in  the  blood.  In  leukaemia,  in  which  a  most  remark- 
able increase  in  the  leucocytes  may  occur,  the  blood  at  times  pre- 
sents a  milky  appearance.  This  is  not  referable  to  any  change  of 
the  normal  coloring-matter,  however,  but  to  increase  of  the  leucocytes 
as  such. 

The  Odor. — The  odor  of  the  blood  is  characteristic,  but  is  different 
in  different  species  of  animals.  It  can  be  intensified  by  treating  the 
blood  with  a  small  amount  of  fairly  concentrated  sulphuric  acid.  In 
part  it  is  owing  to  the  presence  of  odorous  salts  of  certain  fatty 
acids,  and  to  a  slight  degree  also  to  trimethylamin.  Other  sub- 
stances, however,  are  also  concerned  in  its  production,  but  of  their 
nature  nothing  is  known. 

The  taste  of  blood  is  salty,  but  at  the  same  time  insipid.  It  is 
referable,  no  doubt,  to  the  mineral  constituents  of  the  plasma. 

The  Specific  Gravity. — The  specific  gravity  of  normal  blood 
seems  to  vary  with  the  amount  of  haemoglobin.  It  is  influenced 
by  the  age  and  sex  of  the  individual,  the  process  of  digestion,  the 
amount  of  exercise  taken,  pregnancy,  etc.  It  is  dependent,  more- 
over, upon  the  bloodvessel  from  which  it  is  drawn,  and  differs 
somewhat  in  different  animals.  Generally  speaking,  it  varies  in 
healthy  adults  between  1.058  and  1.062.  It  is  higher,  as  a  rule,  in 
men  (1.059)  than  in  women  (1.065)  and  in  children  (1.050-1.052). 
Under  pathological  conditions  the  variations  are  much  greater.  It 
may  thus  fall  to  1.025  and  rise  to  1.068.  It  is  to  be  noted,  more- 
over, that  the  specific  gravity  does  not  necessarily  vary  with  the 
amount  of  haemoglobin,  and  in  nephritis,  various  circulatory  dis- 
turbances, in  leukaemia,  and  in  the  anaemias  following  profuse  hem- 
orrhages or  inanition,  care  should  be  had  not  to  draw  inferences 
as  to  the  amount  of  blood  coloring-matter  from  a  determination  of 
the  specific  gravity. 

Determination  of  the  Specific  Gravity. — Hammerschlag's 
Method. — A  cylinder  measuring  about  10  cm.  in  height  is  partly 
filled  with  a  mixture  of  benzol  (sp.  gr.  0.889)  and  chloroform 
(sp.  gr.  1.526),  so  that  the  specific  gravity  lies  between  1.050 
and  1.060.  Into  this  solution  a  drop  of  blood  is  allowed  to 
fall  directly  from  the  finger,  care  being  taken  that  it  does  not 
come  in  contact  with  the  walls  of  the  vessel.  The  drop,  moreover, 
should  not  be  too  large,  as  it  will  otherwise  separate  into  several 
droplets,  and  thus  give  rise  to  inaccurate  results.  It  is  then  brought 
to  suspension  in  the  middle  of  the  fluid,  by  adding  a  little  chloro- 
form or  ether,  according  to  its  tendency  to  sink  to  the  bottom  or  to 
rise  to  the  surface.  As  soon  as  it  remains  in  the  middle  the  mixture 
is  filtered  through  a  layer  of  linen,  and  its  specific  gravity  deter- 
mined by  means  of  an  accurate  hydrometer,  which  is  graduated  to 
the  fourth   decimal.     The    figure    obtained  represents   the    specific 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  309 

gravity  of  the  blood.  It  is  said  that  no  error  is  incurred  through 
evaporation,  and  the  mixture  may  be  kept  indefinitely. 

The  Amount. — The  total  amount  of  blood  which  is  contained  in 
the  body  corresponds  in  vertebrate  animals  to  from  one-twelfth  to 
one-fourteenth  of  the  body-weight.  It  is  most  conveniently  deter- 
mined according  to  the  following  method  : 

Method  of  Welcker. — From  the  animal  to  be  investigated  10  to 
30  c.c.  of  blood  are  first  withdrawn  and  carefully  defibrinated 
by  whipping.  This  amount  is  weighed  together  with  the  fibrin  and 
set  aside.  The  animal  is  then  bled  to  death  and  the  blood  defibri- 
nated as  before.  After  removal  of  the  feces,  the  intestinal  contents, 
and  the  gall-bladder,  the  entire  body  is  finely  minced  and  repeatedly 
extracted  with  water ;  the  washings  are  added  to  the  large  mass  of 
blood.  The  total  volume  is  now  ascertained  and  the  color  of  the 
bloody  fluid  compared  with  that  of  the  first  10  to  30  c.c,  by 
diluting  this  portion  with  water  until  the  color  of  both  portions 
is  the  same.  From  the  degree  of  dilution,  the  amount  of  blood 
which  is  present  in  the  larger  volume  of  fluid  can  then  readily  be 
determined. 

CHEMICAL  EXAMINATION  OF  THE  BLOOD. 

Reaction. — The  reaction  of  the  blood,  owing  to  the  presence  of 
monosodium  carbonate  and  disodiuni  phosphate,  is  slightly  alkaline. 
This  may  be  demonstrated  by  repeatedly  drawing  a  strip  of  neutral 
litmus-paper  thoroughly  moistened  with  a  concentrated  solution  of 
common  salt  through  the  blood,  and  rapidly  washing  off  the 
corpuscles  with  the  same  solution.  In  man  the  degree  of  alkalinity 
under  normal  conditions  corresponds  to  from  300  to  325  milli- 
grammes of  sodium  hydrate  for  every  100  c.c.  of  blood.  These  figures 
were  obtained  with  Lowy's  method  (see  below),  and  are  higher  than 
those  usually  given  in  text-books,  but  probably  are  more  nearly 
correct. 

Owing  to  the  formation  of  certain  acids  the  alkalinity  of  the 
blood  rapidly  diminishes  after  being  shed,  and  for  this  reason  its 
determination  is  a  Bomewhat  difficult  matter.  Generally  speaking, 
it  is  a  little  lower  in  women  and  children  than  in  men,  and  is 
influenced  to  a  certain  degree  by  the  process  of  digestion,  the 
amount  of  exercise  taken,  etc.  At  the  beginning  of  digestion, 
when  hydrochloric  acid  is  being  seeretcd  in  large  amounts,  it  is  thus 
increased,  while  later  on,  when  the  hydrochloric  acid  and  peptones 
are  reabsorbed,  it  is  diminished.  On  the  whole,  however,  these 
normal  variation-  are  slight.  ( rreater  deviations  have  been  observed 
under  pathological  conditions,  and  are  especially  noted  in  leukaemia, 
pernicious  anaemia,  nephritis,  and  diabetes  when  accompanied  by 
coma,  in  connection  with  high  fever,  during  the  algid  state  of 
Asiatic     cholera,    etc.       It     is    interesting    to    note,    however,    that 

according   to  v.  Limbeck  these  observations  may  be  referable  to 


310  THE  BLOOD. 

faulty  technique,  as  with  his  method  (see  below)  no  difference  could 
be  shown  to  exist  between  normal  and  pathological  conditions.  It  is 
conceivable,  of  course,  that  in  disease  the  alkalinity  of  the  blood 
may  diminish  more  rapidly  after  being  shed  than  under  normal  con- 
ditions, and  this  may  account  for  the  different  results  which  have 
been  reached  with  other  methods.  But,  on  the  other  hand,  v.  Lim- 
beck's method  may  likewise  not  be  free  from  error. 

The  tenacity  with  which  the  living  organism  tends  to  maintain 
the  normal  composition  of  the  bodily  fluid  is,  of  course,  well  known, 
but  that  it  is  not  always  able  to  do  so  is  also  an  established  fact. 
Herbivorous  animals  thus  rapidly  die  when  given  large  amounts  of 
mineral  acids,  and  it  may  be  shown  that  the  alkalinity  of  their  blood  is 
then  markedly  diminished.  In  cases  of  poisoning  with  strychnin, 
arsenious  acid,  carbon  monoxide,  and  amyl  nitrite,  moreover,  where  a 
marked  albuminous  decomposition  occurs  and  lactic  acid  appears  in 
the  urine,  the  same  result  is  obtained.  Carnivorous  animals,  on  the 
other  hand,  are  more  resistant  in  this  respect,  and  will  stand  much 
larger  amounts.     The  result,  however,  is  the  same. 

Lbwy's  Method. — Five  c.c.  of  blood,  obtained  from  one  of  the 
superficial  veins  of  the  arm,  are  allowed  to  flow  into  a  small  flask, 
which  is  provided  with  a  long  and  partially  graduated  neck,  and 
contains  45  c.c.  of  a  0.25  per  cent,  solution  of  ammonium  oxalate. 
Coagulation  is  thus  prevented,  and  the  blood  made  lake-color — i.  e.} 
the  haemoglobin  is  dissolved  from  the  stroma  of  the  red  corpuscles. 
The  mixture  is  then  titrated  with  a  one-twenty -fifth  normal  solution 
of  tartaric  acid,  using  as  an  indicator  lacmoid  paper  which  has  been 
soaked  in  a  concentrated  solution  of  magnesium  sulphate.  The 
number  of  cubic  centimeters  employed  to  neutralize  the  5  c.c.  of 
blood,  multiplied  by  0.0016,  will  then  indicate  the  degree  of  alka- 
linity in  terms  of  sodium  hydrate.  The  percentage  is  obtained  by 
multiplying  the  resulting  figure  by  20. 

v.  Limbeck's  Method. — Ten  c.c.  of  blood  are  allowed  to  flow 
into  200  c.c.  of  boiling  water,  to  which  5  c.c.  of  a  one-tenth  nor- 
mal solution  of  hydrochloric  acid  have  been  added.  The  resulting 
solution,  which  is  clear  and  of  a  brownish  color,  is  now  retitrate^d 
with  a  one-tenth  normal  solution  of  sodium  hydrate,  using  as  indi- 
cator the  syntonin  precipitate  which  occurs  on  neutralization.  The 
difference  between  the  10  c.c.  of  the  hydrochloric  acid  and  the 
sodium  hydrate  solution  is  multiplied  by  0.004.  The  result  indi- 
cates the  alkalinity  of  the  5  c.c.  of  blood,  and  to  obtain  the  per- 
centage this  is  multiplied  by  20. 

The  Chemical  Composition  of  the  Blood,  as  a  Whole. 

As  the  blood  constitutes  the  most  important  channel  through 
which  the  food-stuffs  reach  the  various  tissues  of  the  body,  and 
through  which  waste  matter  is  carried  away,  we  may  expect  to 
find    here    representatives    of  both    classes    of  substances.     This  is 


CHEMICAL  EXAMINATION   OF  THE  BLOOD.  311 

actually  the  case  ;  but  as  the  waste  matter  is  rapidly  eliminated, 
representatives  of  this  group  are  normally  present  in  only  very 
small  amounts.  Certain  food-stuffs,  moreover,  which  are  not  im- 
mediately required  by  the  body  in  large  quantities,  such  as  fats  and 
carbohydrates,  are  likewise  present  only  in  traces.  They  are  stored 
in  various  tissues  of  the  body  until  needed,  but  even  then  only 
small  quantities  appear  in  the  blood  at  one  time.  The  only  food- 
stuffs, in  fact,  which  are  always  present  in  the  blood  in  large  quan- 
tities, are  the  albumins.  These,  however,  must  be  sharply  separated 
into  two  classes,  viz.,  into  those  which  are  normally  present  as 
integral  constituents  of  the  cellular  elements  of  the  blood,  and  which, 
of  course,  do  not  represent  food-material,  and  into  the  so-called  cir- 
culating albumins  of  the  plasma.  In  addition  to  these  elements,  we 
also  meet  with  mineral  salts,  a  very  large  amount  of  water,  and 
with  certain  gases. 

A  general  idea  of  the  chemical  composition  of  human  blood 
may  be  had  from  the  following  table,  which  is  calculated  for  1000 
parts  by  weight : 

Red  corpuscles1 480.00 

Water 276.90 

Oxyhemoglobin 193.90 

Stroma2    .    .    . 9.12 

Plasma 520.00 

Water 477.36 

Albumins 35.88 

Extractives 2.39 

Inorganic  salts 4.36 

From  this  analysis  it  will  be  seen  that  almost  one-half  of  the 
total  weight  of  the  blood  is  referable  to  cellular  elements,  and  that 
in  the  liquid  portion  proper  there  is  not  more  than  8.2  per  cent. 
of  solids,  of  which  6.9  per  cent,  is  albumins,  and  0.84  per  cent, 
mineral  salts  and  0.46  per  cent,  extractives.  The  predominating 
solid  substance  in  the  blood  is  the  oxyhemoglobin  ;  it  represents 
about  19  per  cent,  of  the  total  weight  of  the  blood,  40  per  cent,  of 
tin-  weight  of  the  blood-corpuscles,  and  95  per  cent,  of  all  organic 
material  present. 

The  native  albumins,  which  arc  found  in  the  circulating  blood, 
are  serum-albumin, serum-globulin,  and  fibrinogen.  The  extractives 
comprise  traces  of  fats,  soaps  of  the  higher  fatty  acids,  lecithin, 
glucose,  animal  gum,  glycogen,  sarcolactic  acid,  urea,  kreatin,  uric 
acid,  and  possibly  also  minimal  amounts  of  the  xanthin  bases. 
Nucleo-albumins,  albumoses,  and  some  of  the  lower  fatty  acids,  oxy- 
butyria acid,  acetone,  bilirubin,  melanin,  and  other  less  well-known 
bodies  have  further  been  found  under  pathological  conditions,  but 
are  not   aeen  in  normal    blood. 

I'll"  mineral  constituents  comprise  sodium,  potassium,  calcium, 
magnesium,  and   iron.     With  the  exception  of  the  last  mentioned, 

1  The  white  corpuscle*  becau  cant  In  amount,  have  been  Ignored. 

i     ...  , 


312  THE  BLOOD. 

they  are  present  as  chlorides,  phosphates,  carbonates,  and  to  a  slight 
extent  also  as  fluorides.  Some  of  these  occur  in  the  blood  as  such, 
while  others  form  more  or  less  intimate  combinations  with  the  albu- 
mins. The  iron  largely  occurs  as  an  integral  constituent  of  the 
haemoglobin  molecule,  of  which  it  forms  from  0.39  to  0.47  per  cent. 
Traces  are  also  present  in  certain  leucocytes,  and  notably  those  of  ' 
the  oxyphilic  variety.  In  the  plasma  itself  it  is  at  times  met 
with  in  infinitesimally  small  amounts,  and  is  then  referable  to  the 
destruction  of  leucocytes. 

In  addition  to  these  constituents  of  the  normal  blood,  we  further 
meet  with  certain  gases,  viz.,  oxygen,  carbon  dioxide,  and  nitrogen. 
Of  these,  oxygen  and  carbon  dioxide  occur  partly  in  solution  and 
partly  in  combination  with  haemoglobin,  while  nitrogen  is  found 
only  in  solution.  The  carbon  dioxide,  moreover,  is  in  part  present 
as  a  soluble  bicarbonate,  and  to  a  certain  extent  also  in  combina- 
tion with  the  albumins  of  the  plasma.  These  gases  may  be  ex- 
tracted from  the  blood  in  their  entirety  by  exposure  to  a  vacuum.  As 
the  nitrogen  is  simply  held  in  solution,  its  volume  is  constant,  and 
corresponds  to  2  per  cent,  by  volume  no  matter  whether  the  blood 
is  obtained  from  an  artery  or  a  vein.  The  relative  amount  of  oxy- 
gen and  carbon  dioxide,  on  the  other  hand,  is  subject  to  great  varia- 
tions. From  the  arterial  blood  of  dogs  it  is  thus  possible  to  obtain 
21  per  cent,  of  oxygen  by  volume,  and  38  per  cent,  of  carbon  di- 
oxide, while  venous  blood  contains  as  much  as  46  percent,  of  carbon 
dioxide  and  only  12  per  cent,  of  oxygen. 

As  these  gases  can  be  obtained  by  exposure  to  a  vacuum,  it  follows 
that  their  combination  with  oxyhemoglobin  cannot  be  very  strong ; 
it  is  surprising,  however,  to  note  that  in  this  manner  not  only  that 
portion  of  the  carbon  dioxide  is  obtained  which  is  in  combination 
with  albuminous  material,  but  also  the  carbon  dioxide  of  the  car- 
bonates. This  phenomenon  is  owing  to  the  fact  that  in  conse- 
quence of  the  vacuum  the  red  corpuscles  are  broken  down,  and 
that  the  haemoglobin  which  is  thus  set  free  is  then  capable  of  exer- 
cising  its  acid  properties,   and  causes  decomposition    of   the  salts. 

The  Plasma. 

In  order  to  obtain  blood-plasma  it  is  necessary  to  prevent  coagu- 
lation of  the  blood.  This  may  be  accomplished  in  various  ways. 
It  has  thus  been  found  that  following  the  intravenous  injection  of 
certain  albumoses  (peptones),  or  of  an  infusion  of  the  mouth  parts 
of  the  officinal  leech,  as  also  after  ligation  of  the  bloodvessels  of 
the  liver  and  intestines,  the  blood  remains  liquid  after  being 
shed.  On  allowing  it  to  stand  at  a  low  temperature  the  blood- 
corpuscles  settle  to  the  bottom,  when  the  supernatant  fluid  may 
be  siphoned  off;  or  the  blood  may  be  centrifugalized  at  once  and 
separation  of  the  cellular  elements  effected  in  this  way.  Blood- 
plasma  that  has  been   obtained  after  the   injection  of  albumoses  is 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  313 

termed  albumose-plasma,  or,  loss  correctly,  peptone-plasma,  in  con- 
tradistinction t<>  saltrjplasma,  which  results  when  blood  is  received  in 
a  solution  of  a  neutral  salt,  whereby  coagulation  is  also  prevented. 
To  this  end  it  is  best  to  employ  a  saturated  solution  of  sodium 
sulphate  or  a  10  per  cent,  solution  of  sodium  chloride,  to  which  the 
blood  i-  added  in  an  equal  amount.  A  saturated  solution  of  inagne- 
sium  sulphate  may  likewise  be  used,  in  the  proportion  of  one  part 
for  three  parts  of  blood,  but  is  not  so  satisfactory,  as  it  causes  pre- 
cipitation of  certain  albumins  which  are  essential  to  coagulation. 
After  standing  tor  twenty-four  hours  the  plasma  may  be  siphoned 
off,  or  may  lie  separated  from  the  corpuscles  at  once  by  centrifugation. 

As  coagulation  of  the  blood  is  apparently  dependent  upon  the 
presence  of  soluble  calcium  salts,  coagulation  may  also  be  prevented 
by  precipitating  with  ammonium  oxalate  those  which  are  present  in 
the  blood.  To  this  end  the  blood  is  received  in  a  solution  of  am- 
monium oxalate,  such  that  the  quantity  of  the  latter  present  in 
the  mixture  amounts  to  about  0.1  per  cent.  This  constitutes 
oxalate-plasma. 

Of  especial  value  in  these  examinations  is  the  blood  of  the  horse, 
in  which  coagulation  occurs  much  more  slowly  than  in  that  of 
mammals.  If  this  is  available,  it  is  only  necessary  to  receive  it  in 
a  narrow  cylinder  surrounded  with  a  freezing-mixture.  Kept  in 
this  manner  it   will  remain  liquid  for  several  days. 

Separated  from  the  corpuscles,  the  plasma  is  a  clear,  straw-colored, 
slightly  viscid  fluid,  of  alkaline  reaction,  and  a  specific  gravity 
varying  between  1.026  and  1.029  in  man.  It  is  capable  of  under- 
going coagulation,  like  the  native  blood,  and  is  thus  converted  into 
blood-serum.  Its  general  chemical  composition  has  already  been 
considered.  It  contains  but  8.2  per  cent,  of  solids,  of  which  6.9 
per  cent,  is  represented  by  albumins.  These  are  serum-albumin, 
serum-fflobulin,  and  fibrinogen.  The  relation  between  these  bodies 
i-  subject  to  cousiderable  variations.  In  all  animals,  however,  the 
globulins  predominate,  and  in  some  indeed,  as  in  snakes,  serum- 
albumin  is  apparently  absent.  In  the  horse  the  globulins  con- 
stitute about  64.6  per  cent,  of  the  total  amount  of  albumins.  In 
1000  parts  by  weight  ilanmiarsten  thus  found  38.4  parts  of  serum- 
globulin,  6.5  parts  of  fibrinogen,  and  24.6  parts  of  serum-albumin. 
Of  these  albumins,  fibrinogen  is  of  especial  interest,  as  it  represents 
the  mother-substance  of  fibrin,  and  is  thus  intimately  connected  with 
the  process  of  coagulation. 

Fibrinogen. — Isolation.  —  Fibrinogen  is  most  conveniently  ob- 
tained  from  the  plasma  by  half-saturation  with  sodium  chloride — 
i.  e.f  by  treating  oik;  volume  of  the  plasma  with  an  equal  volume  of 
a  saturated  solution  of  common  salt.     The  resulting  precipitate  of 

fibrinogen    is    filtered    off,  washed  with    a    half-saturated    solution  of 

sodium  chloride,  and  dissolved  in  an  8  per  cent,  solution  of  the  salt. 

To  further  purify  the  Bubstance,  this  solution  is reprecipitated,  redis- 

ed,  and   the  process   repeated   twice.     The   i\w.\\  precipitate   is 


314  '  THE  BLOOD. 

pressed  between  filter-paper  and  suspended  in  water,  in  which  it 
readily  dissolves  owing  to  the  small  amount  of  salt  that  still  remains. 
This  may  be  removed  by  dialysis.  The  purified  substance,  in  a  moist 
state,  appears  in  the  form  of  white  flocculi,  which  readily  coalesce  to 
form  a  tough  elastic  mass. 

The  isolation  of  the  fibrinogen  must  be  performed  rapidly,  as  pro- 
longed exposure  to  the  half-saturated  salt  solution  tends  to  render 
the  substance  insoluble. 

Properties. — Fibrinogen  belongs  to  the  class  of  globulins.  It  is 
insoluble  in  distilled  water,  but  soluble  in  dilute  solutions  of  the 
neutral  salts.  From  these  solutions  it  may  be  precipitated  by  dial- 
ysis, by  increasing  the  amount  of  the  salt,  and  by  passing  a  stream  of 
carbon  dioxide  through  the  solution.  When  kept  under  water  for  a 
comparatively  short  time  it  is  rendered  insoluble.  When  heated  to 
56°  C.  coagulation  occurs,  but  it  appears  that  the  fibrinogen  is  at 
the  same  time  decomposed  into  two  other  globulins,  one  of  which 
coagulates  at  the  temperature  just  mentioned,  while  the  other 
remains  in  solution  until  the  temperature  reaches  65°  C.  Of  the 
nature  of  these  two  substances,  however,  we  know  but  little ;  it  is 
possible  that  one  is  the  so-called  fibrinoglobulin,  which,  as  we  shall 
see  later,  is  formed  during  coagulation  of  the  blood.  Fibrinogen 
turns  the  plane  of  polarized  light  to  the  left  ;  its  rotation  for  the 
yellow  D  line  corresponds  to  — 52.5  degrees.  It  consists  of  carbon, 
hydrogen,  nitrogen,  sulphur,  and  oxygen,  in  the  proportion  of  52.93, 
6.9,  16.6,  1.25,  and  22.26,  respectively.  Its  most  characteristic 
property  is  its  tendency  to  the  formation  of  fibrin,  and  upon  this  its 
specific  test  and  quantitative  estimation  are  based.  This  transforma- 
tion will  be  considered  in  detail  later  (see  Coagulation). 

In  addition  to  the  blood-plasma,  fibrinogen  has  been  found  in  the 
chyle,  the  lymph,  and  various  exudates  and  transudates. 

Serum-globulin. — This  substance  has  also  been  termed  para- 
globulin,  Alexander  Schmidt's  fibrinoplastic  substance,  and  serum- 
casein.  Like  fibrinogen,  it  is  found  in  the  plasma  of  the  blood,  in 
the  lymph,  in  various  exudates  and  transudates ;  but  it  likewise 
occurs  in  the  serum,  in  the  white  and  red  corpuscles  of  the  blood, 
and  in  traces  at  least  in  all  cellular  elements  of  the  animal  body. 
In  the  urine  it  has  been  encountered  in  association  with  serum- 
albumin  under  various  pathological  conditions. 

Isolation. — Serum-globulin  is  most  conveniently  obtained  from 
blood-serum  by  half-saturation  with  ammonium  sulphate — i.  e.,  by 
treating  a  given  volume  of  the  serum  with  the  same  amount  of  a 
saturated  solution  of  the  salt.  Saturation  with  magnesium  sulphate 
in  substance  may  also  be  employed.  In  either  case  the  precipitated 
serum-globulin  is  filtered  off,  washed  with  the  corresponding  salt 
solution,  dried  at  115°  C,  then  washed  with  boiling  water  to  remove 
the  remaining  salts,  extracted  with  alcohol,  then  with  ether,  and 
finally  dried  and  weighed.  In  any  case  the  original  solution  should 
be  nearly  neutral  in  reaction. 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  315 

Properties. — As  the  term  indicates,  serum-globulin  belongs  to  the 
class  of  globulins.  In  its  moist  state  it  represents  a  snowy-white, 
finelv  flocculent  mass,  which  is  not  tough  and  elastic  like  fibrinogen. 
From  its  solutions  the  substance  can  be  precipitated,  as  already  in- 
dicated, by  saturation  with  magnesium  sulphate  or  by  half-saturation 
with  ammonium  sulphate.  Sodium  chloride  causes  only  an  incom- 
plete separation  of  the  substance  when  added  to  saturation,  while 
half-saturation  is  of  no  effect  at  all.  It  is  thus  an  easy  matter  to 
isolate  serum-globulin  when  fibrinogen  is  present.  An  incomplete 
separation  also  occurs  when  its  neutral  or  feebly  acid  solutions  (using 
acetic  acid)  are  diluted  from  ten  to  twenty  times  with  distilled 
water,  or  by  passing  a  stream  of  carbon  dioxide  through  such  dilute 
solutions.  In  the  presence  of  from  5  to  10  per  cent,  of  sodium  chlo- 
ride its  solutions  coagulate  at  75°  C.  They  cause  a  rotation  of  the 
polarized  light  to  the  left,  the  specific  degree  corresponding  to  47.8. 

According  to  Morner,  serum-globulin  yields  a  reducing  substance 
when  boiled  with  dilute  mineral  acids.  It  might  thus  be  supposed 
that  serum-globulin  is  in  reality  a  glucoproteid,  and  not  a  natiye 
albumin,  but  it  is  possible  also  that  the  reaction  is  due  to  the  acci- 
dental presence  of  a  glucoproteid,  which  is  thrown  down  together 
with  the  globulin. 

Though  serum-globulin  is  usually  spoken  of  as  a  unity — that  is, 
an  individual  substance — it  appears  likely  that  the  compound  which 
is  thrown  down  upon  saturation  with  magnesium  sulphate  repre- 
sents a  mixture  of  seyeral  globulins.  If  this  material  is  thus  dis- 
solved in  dilute  saline  solution  and  subjected  to  dialysis,  a  precipitate 
forms  which  possesses  all  the  properties  of  serum-globulin  which  we 
regard  as  characteristic  of  globulins.  The  remaining  solution,  how- 
ever, contains  an  albuminous  substance  which  may  be  thrown  down 
by  magnesium  sulphate,  and  which  when  isolated  in  this  manner 
differs  only  from  the  first  precipitate  in  being  soluble  in  water. 
Formerly  it  was  thought  that  all  globulins  are  insoluble  in  water, 
but  it  is  thus  shown  that  at  least  one  form  differs  in  this  respect. 

The  globulin  which  is  obtained  from  blood-plasma  is  also  spoken 
of  as  plasma-globulin,  in  contradistinction  to  the  so-called  cell- 
globulin;  and  Bammarsten's  secondary  globulins,  which  are  found 
in  the  serum  together  with  plasma-globulin,  are  thought  to  result 
from  disintegration  of  the  leucocytes  and  the  fibrinogen  molecule, 
respectively.  A.ccordingly  we  also  find  more  serum-globulin  in  the 
--'Turn  1 1 1 : m  in  tin-  plasma.  Chemically,  these  various  globulins  are 
not  sufficiently  characterized  to  warrant  a  separate  description,  and 
as  they  are  all  thrown  down  together  with  our  present  methods  of 
isolation,  it  follows  that  the  numerical  data  regarding  the  elementary 
composition  of  serum-globulin  as  unity  must  also  be  more  of  less 
at  fault.  We  find,  a-  a  matter  ,,{'  fact,  that  these  data  are  by  no 
mean-  constant.  The  value  of  carbon  thus  varies  between  52.32  and 
53.3,  and  that  of  nitrogen  between  15.61  and  16.25,  which  would 
represent  a  difference  of  nearly  1  and  0.64  per  cent.,  respectively 


316  THE  BLOOD. 

— that  is,  amounts  which  could  scarcely  be  owing  to  technical 
errors. 

Serum-albumin. — Serum-albumin  is  found  in  the  plasma,  the 
serum,  the  lymph,  in  exudates  and  transudates,  and  under  certain 
pathological  conditions  also  in  the  urine,  where  it  usually  occurs  in 
association  with  serum-globulin.  It  is  most  conveniently  obtained 
from  blood-serum  after  removal  of  the  serum-globulin  by  saturation 
with  magnesium  sulphate  at  a  temperature  of  30°  C.  The  nitrate  is 
saturated  with  sodium  sulphate  or  ammonium  sulphate  at  40°  C,  or 
treated  with  acetic  acid,  so  that  the  solution  contains  about  1  per 
cent.  In  either  case  the  precipitated  serum-albumin  is  filtered  off, 
pressed  between  layers  of  filter-paper,  dissolved  in  water  (the  reac- 
tion should  be  neutral),  and  separated  from  the  remaining  salt  by 
dialysis.  From  its  aqueous  solutions  it  is  finally  obtained  by 
evaporation  at  a  low  temperature  or  by  precipitation  with  alcohol, 
which  must  be  rapidly  removed,  however,  as  otherwise  it  will  cause 
coagulation  of  the  albumin. 

In  the  dry  state  serum-albumin  is  a  transparent,  gum-like,  brittle, 
hygroscopic  mass,  or  a  white  powder,  which  can  be  heated  to  100°  C. 
without  undergoing  decomposition.  Solutions  of  the  pure  substance 
in  distilled  water  coagulate  at  50°  C,  while  in  the  presence  of  salts 
a  higher  temperature  is  necessary.  This  varies  with  the  amount  of 
salt  present,  as  also  with  the  concentration  of  the  albumin.  A  1 
to  2  per  cent,  solution  containing  5  per  cent,  of  sodium  chloride 
coagulates  between  75°  and  90°  C.  From  its  salt  solutions  serum- 
albumin  may  be  obtained  in  crystalline  form.  Its  specific  rotation 
in  distilled  water  varies  between  62.6°  and  64.6° — a.  [D]. 

According  to  Halliburton,  the  serum-albumin  of  mammalian 
blood-serum  is  not  a  single  substance,  but  consists  of  three  distinct 
albumins,  which  he  terms  a-,  /?-,  and  ^-serum-albumin.  They  are 
said  to  coagulate  at  73°  C,  77°  C,  and  84°  C,  respectively.  In 
cold-blooded  animals,  a-serum-albumin  only  is  said  to  occur. 

Separation  of  the  Albumins  of  the  Blood-plasma  from  Each 
Other. — To  isolate  the  fibrinogen,  the  plasma  is  treated  with  an 
equal  volume  of  a  saturated  solution  of  sodium  chloride.  The  result- 
ing precipitate  is  filtered  off  and  purified  as  described.  The  filtrate 
contains  serum-globulin  and  serum-albumin.  The  globulin  is  pre- 
cipitated by  saturation  with  magnesium  sulphate,  filtered  off,  and 
likewise  purified.  The  filtrate  contains  only  the  serum-albumin, 
which  may  be  obtained,  as  just  described,  by  saturation  with  sodium 
or  ammonium  sulphate. 

Quantitative  Estimation  of  the  Total  Albumin  of  the  Plasma. 
— The  albumins  are  most  conveniently  estimated  by  treating  a  care- 
fully measured  and  weighed  amount  of  the  plasma,  after  neutraliza- 
tion with  acetic  acid,  with  five  times  its  volume  of  alcohol.  After 
standing  for  twenty-four  hours  the  solution  is  boiled  for  several 
minutes,  and  the  resulting  precipitate  collected  on  a  weighed  filter, 
washed  with  hot  alcohol,  then  with  ether,  dried  at  115°  C,  weighed, 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  317 

and  incinerated.  The  weight  of  the  ash  is  deducted  from  the  weight 
of  the  precipitate. 

More  accurate  is  the  following  method  :  a  carefully  measured 
and  weighed  amount  of  the  plasma  is  treated  with  one-half  its  volume 
of  a  saturated  solution  of  sodium  chloride  and  a  slight  excess  of 
tannic  acid.  In  the  resulting  precipitate  the  nitrogen  is  then  esti- 
mated according  to  Kjeldahl's  method.  When  multiplied  by  6.37 
the  corresponding  amount  of  albumin  is  obtained. 

To  remove  all  albumins  from  the  blood,  Cavazzani's  method  may 
be  employed.  To  this  end,  20-30  c.c.  of  blood  are  added  to  200  c.c. 
of  distilled  water,  and  treated  with  five  or  six  drops  of  a  solution 
consisting  of  ten  parts  of  acetic  acid  (sp.  gr.  1.040)  and  one  part  of 
lactic  acid.  The  mixture  is  boiled  for  about  ten  minutes,  filtered, 
and  the  precipitate  washed  separately  with  hot  water,  and  finally 
pressed  in  a  piece  of  muslin.  The  filtrate  and  washings,  which  are 
practically  colorless,  are  then  concentrated  to  a  small  volume.  Any 
traces  of  albumin  which  may  still  be  present  thus  separate  out  and 
are  filtered  off.  If  too  much  of  the  acid  solution  has  been  added,  the 
mixture  may  not  clear  on  boiling.  In  that  event  a  few  crystals  of 
sodium  carbonate  are  added,  when  coagulation  promptly  occurs.  On 
the  other  hand,  it  may  at  times  be  necessary  to  add  a  few  drops 
more  of  the  acid  solution. 

The  remaining  constituents  of  the  plasma  are  also  found  in  the 
serum,  and  will  be  considered  in  that  connection. 

The   Serum. 

The  serum  results  from  the  blood-plasma  during  the  process  of 
coagulation.  It  is  most  conveniently  obtained  by  whipping  blood 
immediately  after  being  shed,  whereby  the  greater  portion  of  the 
fibrin  is  removed  and  the  formation  of  large  clots  prevented.  The 
corpuscles  and  smaller  pieces  of  fibrin  are  separated  by  centrifuga- 
tion  or  by  allowing  the  fluid  to  stand  in  the  cold  until  sedimen- 
tation has  occurred.  The  serum  is  then  siphoned  off  and  filtered. 
It  thus  appears  as  a  slightly  viscid,  fairly  transparent  fluid  of  a  light 
straw  color,  which  presents  a  feebly  alkaline  reaction  and  a  specific 
gravity  varying  between  1.026  and  1.029  in  man.  In  its  chemical 
composition  serum  differs  from  plasma  principally  in  the  presence 
of  the  fibrin  ferment  and  in  the  absence  of  fibrinogen.  In  its  place, 
however,  traces  of  two  other  globulins,  which  arc  not  present  in  the 
plasma,  arc  found.  One  of  these  is  termed  fibrinoglobulin,  and  is 
thought  to  result  during  the  formation  of  fibrin  from  fibrinogen. 
The  .,ther  ifl  the   BO-Called    Cell-globulin,  and    i<   supposedly   referable 

to  the  decomposition  of  leucocytes  during  the  process  of  coagulation. 
The  remaining  constituents  are  qualitatively  the  same  in  both  fluids. 
Slight  quantitative  differences,  however,  exist.  A  portion  of  the 
calcium,    magnesium,    and    phosphoric   acid     is    thus    eliminated 


318  THE  BLOOD. 

together  with  the  fibrin,    and  accordingly  lower  values  are  found 
in  the  serum  than  in  the  plasma. 

An  idea  of  the  mineral  constituents  of  the  serum,  and  their 
quantitative  relations,  may  be  had  from  the  accompanying  table  : 

Man. 

Potassium  oxide 0.387 — 0.401  pro  mille. 

Sodium  oxide 4.290  " 

Chlorine      3.565—3.659  " 

Calcium  oxide 0.155  " 

Magnesium  oxide      0.101  " 

From  this  it  will  be  seen  that  sodium  in  the  form  of  the  chloride 
largely  predominates  in  the  serum,  while  potassium  occurs  only  in 
small  amounts.  This  is  exactly  the  reverse  of  what  is  seen  in  the 
morphological  elements  of  the  body,  of  which  potassium  compounds 
are  the  principal  salts  present.  It  is  noteworthy,  moreover,  that  the 
amount  of  sodium  chloride  is  practically  constant  in  the  blood,  no 
matter  whether  large  quantities  are  ingested  or  the  salt  is  given  in  only 
small  amounts.  During  starvation  even,  or  when  the  potassium  salt 
is  artificially  substituted,  the  amount  present  in  the  blood  remains 
practically  constant.  Apparently  it  occurs  only  in  solution,  and  does 
not  form  an  integral  part  of  the  albuminous  molecule,  as  is  the  case 
with  the  phosphates  of  the  blood.  Of  these,  traces  only  are  present 
in  solution,  while  the  greater  portion  is  more  or  less  intimately  com- 
bined with  the  albumins.  As  the  lecithins  which  are  found  in  the 
blood  also  contain  phosphorus,  it  follows  that  these  figures,  which  are 
obtained  by  incinerating  a  given  amount  of  serum  or  plasma  and 
determining  the  phosphoric  acid  in  the  ash,  are  too  high.  In  serum 
which  had  been  freed  from  lecithin  Sertoli  and  Mroczkowski  found 
amounts  varying  between  0.02  and  0.09  pro  mille,  calculated  as  di- 
sodium  phosphate.  In  the  estimation  of  the  sulphates  we  meet 
with  still  greater  difficulties,  as  the  sulphur  of  the  albumins  is 
included  in  the  determination.  The  amount  which  is  present  in 
solution,  however,  is  certainly  very  small.  The  iron  which  is  at 
times  met  with  in  the  serum  is  unquestionably  derived  from  the 
leucocytes,  and  is  an  accidental  constituent.  The  amount  which 
may  be  obtained  is  always  exceedingly  small. 

Of  other  elements,  traces  of  silicon,  fluorine,  copper,  and  man- 
ganese have  at  times  been  observed. 

The  coloring-matter  of  the  serum  and  plasma  is  supposedly  due 
to  a  substance  belonging  to  the  class  of  lipochromes  or  luteins. 

The  albumins  of  the  serum  which  also  occur  in  the  plasma,  viz., 
serum-albumin  and  serum-globulin,  have  been  considered.  Of  the 
fibrinoglobulin  which  is  formed  during  coagulation  of  the  blood, 
and  which  is  thought  to  result  from  decomposition  of  fibrinogen, 
comparatively  little  is  known.  It  coagulates  at  64°  C,  and  appar- 
ently represents  about  one-third  of  the  fibrinogen  molecule.  Of  the 
so-called  cell-globulin,  still  less  is  known,  and  both  are  found  only 
in  traces.     More  important  is  the  presence  of  the  fibrin  ferment, 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  319 

which  is  considered  at  this  place,  as  it  is  usually  regarded  as  an 
albuminous  substance. 

The  Fibrin  Ferment. — The  fibrin  ferment,  or  thrombin  of  Alex- 
ander Schmidt,  is  thought  to  result  from  decomposition  of  the  cellu- 
lar elements  of  the  blood,  and  notably  the  leucocytes  and  blood- 
plates,  in  which  it  is  supposedly  present  in  the  form  of  a  pro- 
enzvme,  the  so-called  prothrombin  of  Alexander  Schmidt.  As  a 
matter  of  fact,  it  is  possible  to  prevent  the  formation  of  the  ferment 
by  allowing  blood  to  flow  directly  from  the  vessel  into  a  solution  of 
one  of  the  neutral  salts.  In  the  circulating  blood  the  ferment  is 
manifestly  not  present,  as  its  solution  when  injected  into  the  blood- 
vessels of  a  living  animal  will  cause  almost  instantaneous  death 
from  thrombosis. 

Isolation. — The  isolation  of  the  fibrin  ferment  is  most  conveni- 
ently accomplished  in  the  following  manner:  taking  the  serum  of  the 
ox,  the  globulins  are  first  precipitated  by  saturation  with  magnesium 
sulphate.  The  filtrate  is  then  diluted  with  water,  and  treated  while 
stirring  with  a  very  dilute  solution  of  sodium  hydrate  until  an 
abundant  and  flocculent  precipitation  of  magnesium  hydroxide  has 
been  brought  about.  This  precipitate,  which  contains  a  large  pro- 
portion of  the  ferment,  is  washed  with  water,  pressed  between  filter- 
paper,  and  dissolved  in  water  by  neutralizing  the  solution  with 
diluted  acetic  acid.  The  salts  are  then  removed  by  dialysis,  when 
the  ferment  can  be  precipitated  by  a  suitable  addition  of  acetic  acid. 

Properties. — Of  the  nature  of  the  product  which  can  thus  be  ob- 
tained little  is  known.  By  some  observers  it  is  regarded  as  a  globu- 
lin, while  others  class  it  as  a  nucleo-albumin.  On  digestion  with 
pepsin  it  is  said  to  yield  a  nuclein  or  a  pseudonuclein.  As  is  the 
case  witli  all  ferments,  its  solutions  are  rendered  inactive  by  exposure 
to  a  moderately  high  temperature  (70°-75°  C),  while  the  various 
antiseptic  substances  which  do  not  interfere  with  the  activity  of 
other  ferments  are  likewise  without  effect  upon  the  fibrin  ferment. 
It-  specific  activity  is  manifested  when  brought  into  contact  with 
fibrinogen,  which  is  apparently  decomposed  by  hydrolysis  into  fibrin 
and  fibrinoglobulin.  To  elfect  this  change,  however,  the  presence 
of  a  neutral  -alt  anil  of  a  soluble  calcium  salt  is  essential.  In  their 
absence  coagulation  does  not  take  place.  According  to  Pekelharing, 
the  fibrin  ferment  is  a  calcium  compound  of  the  pro-enzvme,  and  it 
is  supposed  that  during  the  process  of  coagulation  the  calcium  is 
transferred  to  the  fibrinogen,  whereby  this  is  transformed  in  part 
into  the  insoluble  calcium-containing  fibrin.  At  the  same  time  the 
ferment  is  retransformed  into  its  pro-enzyme,  which  again  combines 
with  calcium  to  form  the  ferment  ;  this,  in  turn, deposits  its  calcium 
on  the  fibrinogen,  and  is  thus  changed  back  into  the  pro-enzyme,  and 
BO  on. 

Fibrin.  —  Fibrin  is  formed  during  the  spontaneous  coagulation  of 
all  albuminous  solutions  which  contain  fibrinogen  and  cellular  ele- 
ment- that  can  give  rise  to  the  fibrin  ferment.     It  is  most  conveni- 


320  THE  BLOOD. 

ently  obtained  by  whipping  freshly  shed  blood  with  a  suitable  instru- 
ment, when  the  fibrin  is  deposited  as  an  elastic,  stringy  material, 
which  may  be  freed  from  adhering  corpuscles  by  thorough  washing 
and  kneading  in  running  water.  Such  fibrin,  however,  is  still  con- 
taminated with  serum-globulin  and  certain  phosphorus-containing 
substances  which  have  resulted  from  the  decomposition  of  leucocytes. 
The  serum-globulin  may  be  removed  by  separate  washing  and  knead- 
ing in  a  5  per  cent,  solution  of  common  salt ;  but  the  other  products, 
as  well  as  the  remains  of  the  corpuscles  of  the  blood,  can  scarcely  be 
removed.  To  obtain  pure  fibrin,  therefore,  it  is  necessary  to  start 
with  filtered  plasma  or  with  filtered  transudates,  which  are  beaten 
with  a  piece  of  whalebone,  after  adding  a  little  serum,  if  the  fluid 
is  not  spontaneously  coagulable.  The  resulting  material  is  washed 
with  water,  then  with  a  5  per  cent,  solution  of  sodium  chloride,  and 
finally  extracted  with  alcohol  and  ether. 

The  fibrin  then  appears  as  a  white  stringy  substance,  which  is 
somewhat  elastic,  but  is  easily  rendered  brittle  on  contact  with 
alcohol  or  on  warming  the  substance  in  water  to  a  temperature  of 
75°  C.  It  is  closely  related  to  the  coagulated  albumins,  and  ac- 
cordingly is  soluble  only  with  difficulty.  It  is  questionable,  more- 
over, whether  solution  of  the  substance  can  be  accomplished  without 
causing  its  decomposition.  If  fibrin  is  thus  placed  in  a  5  to  10  per 
cent,  solution  of  sodium  chloride,  or  a  6  per  cent,  solution  of  sodium 
nitrate,  and  kept  at  a  temperature  of  40°  C,  it  first  swells  up  and 
gradually  disappears  as  such.  In  its  place  two  globulins  or  related 
substances  are  then  said  to  be  found.  This  transformation,  accord- 
ing to  some  observers,  is  referable  to  adherent  bacterial  enzymes. 
In  dilute  alkalies  and  acids  it  likewise  dissolves.  Stronger  acids, 
as  also  the  proteolytic  ferments,  dissolve  the  fibrin,  but  at  the 
same  time  cause  its  transformation  into  acid  albumin  and  albumoses. 
In  water,  alcohol,  and  ether  it  is  entirely  insoluble. 

The  elementary  analysis  of  fibrin  gives  52.68  parts  of  carbon, 
6.83  of  hydrogen,  16.41  of  nitrogen,  1.1  of  sulphur,  and  22.48 
of  oxygen.  It  is  to  be  noted,  further,  that  in  addition  to  these 
elements  calcium  is  constantly  present,  and,  as  has  been  seen,  its 
formation  is  largely  dependent  upon  the  presence  of  a  soluble 
calcium  salt. 

According  to  Lilienfeld,  the  fibrin  is  not  formed  directly  during 
the  decomposition  of  fibrinogen,  but  in  its  stead  we  obtain  another 
substance,  thrombosin,  which  as  an  alkali  compound  is  soluble  in 
water,  but  immediately  combines  with  calcium,  and  then  sejuarates 
out  as  calcium  thrombosin  or  insoluble  fibrin.  This  idea,  however, 
is  not  generally  accepted,  and  it  is  thought  to  be  more  likely,  as 
Pekelharing  suggests,  that  the  calcium  merely  transforms  the  pro- 
enzyme into  the  fibrin  ferment  proper,  and  is  then  mechanically 
carried  down  during  the  separation  of  the  fibrin. 

The  amount  of  fibrin  which  may  be  obtained  from  the  blood,  not- 
withstanding its  bulk,  does  not  exceed  0.1-0.4  per  cent. 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  321 

Estimation. — In  order  to  determine  the  amount  of  fibrin  in  a 
given  volume  of  blood,  from  30  to  40  c.c.  are  placed  in  a  previously- 
weighed  I  (Laker,  which  is  closed  with  an  India-rubber  cap.  Through 
the  centre  of  this  passes  a  piece  of  whalebone  that  is  firmly  fixed 
and  provided  with  a  rudder-like  end,  which  dips  into  the  blood. 
This  is  now  defibrinated  by  beating  with  the  whalebone  paddle, 
when  the  beaker  is  again  weighed  with  its  contents.  The  differ- 
ence, as  compared  with  the  first  weight,  indicates  the  weight  of  the 
blood.  The  beaker  is  then  filled  with  water  and  the  mixture  again 
beaten.  The  fibrin  is  allowed  to  settle,  and  after  being  washed  by 
decantation  with  normal  salt  solution  it  is  collected  on  a  filter  of 
known  weight.  On  this  it  is  further  washed  with  normal  salt  solu- 
tion until  free  from  coloring-matter;  it  is  then  extracted  with  boiling 
alcohol,  then  with  ether,  and  finally  dried  at  115°  C.  and  weighed. 

Other  albumins  in  addition  to  those  already  considered  are  not 
found  in  normal  serum.  Under  pathological  conditions,  however, 
nueleo-albumins  and  deutero-albumoses  may  be  encountered.  They 
will  be  considered  in  future  chapters. 

The  remaining  solids  which  are  found  in  both  plasma  and  serum 
are,  as  has  been  pointed  out,  present  in  only  very  small  amounts. 
The  most  important  of  these  is  glucose.  Its  amount  varies  between 
1  and  1.5  pro  mille,  and  is  but  little  influenced  by  the  character  of 
the  food  unless  a  large  excess  of  carbohvdrates  has  been  ingested. 
In  such  an  event  the  amount  may  increase  to  3  pro  mille,  or  even 
higher,  but  it  then  appears  also  in  the  urine,  whereby  a  further 
increase  is  prevented.  Larger  amounts,  such  as  9  pro  mille,  are 
found  only  under  pathological  conditions. 

It  is  interesting  to  note  that  after  blood  has  been  shed  the  glucose 
rapidly  diminishes  in  amount.  This  phenomenon  is  thought  by 
some  to  be  due  to  the  action  of  a  special  ferment,  the  c/lucolytic 
ferment  of  Lepine.  It  is  supposedly  derived  from  the  leucocytes, 
and,  according  to  Lepine,  is  eliminated  into  the  blood  by  the  pan- 
ereas.  Arthus  and  others  regard  this  process  of  glucolysis  as  a 
post-mortem  change,  but  there  is  reason  to  suppose  that  the  activity 
of  the  glueolytic  ferment  is  exercised  also  during  the  life  of  the 
animal,  and  is  possibly  of  fundamental  importance  in  the  metabo- 
lism of  glycogen.  Whether  Lepine's  ferment  is  identical  with 
another  ferment  which  was  found  in  the  blood,  and  has  been  studied 
by  Rdhmann  and  Bial,  and  which  is  said  to  transform  starch  and 
glycogen  into  glucose,  remains  to  be  seen. 

In  addition  to  glucose,  another  reducing  substance  is  found  in  the 
blood,  which  to  a  certain  degree  is  fermentable  and  is  soluble  in 
ether.  From  the-  researches  of  P.  .Mayer,  it  appears  that  this  sub- 
stance is  a  conjugate  glucuronate.  The  presence  of  jecorin,  on  the 
other   hand,  which    has    repeatedly    been    reported,  is  doubtful. 

.  1  nimalgum  has  also  been  found  in  small  amounts  (0.01 5  per  cent.). 
Glycogen. — Glycogen  is  constantly  present  in  normal  blood.    Its 

21 


322  THE  BLOOD. 

amount,  however,  is  subject  to  great  variations.  As  a  general  rule, 
traces  only  are  found,  but  it  may  increase  at  times  to  1.56  per  cent., 
as  calculated  for  the  blood  as  a  whole.  Larger  amounts  are  seen 
under  pathological  conditions. 

Fat  is  normally  found  to  the  extent  of  from  0.2  to  0.3  per  cent., 
but  may  be  greatly  increased  by  the  ingestion  of  much  fatty  food, 
as  also  in  various  pathological  conditions. 

Urea  is  likewise  found  in  only  very  small  amounts  under  normal 
conditions  (0.016  to  0.020  per  cent.),  while  in  disease  much  greater 
quantities  may  be  encountered.  Ammonia  is  said  to  be  present  in 
normal  blood  to  the  amount  of  0.001  per  cent. 

The  further  occurrence  in  the  blood  of  soaps,  cholesterin,  and 
lecithins,  as  also  of  uric  acid,  kreatin,  carbaminic  acid,  paralactic 
acid,  hippuric  acid,  etc.,  has  been  mentioned.  All  these  bodies  are 
found  in  only  extremely  small  amounts,  and  need  not  be  con- 
sidered at  this  place.  The  pathological  constituents  of  blood,  such 
as  leucin,  tyrosin,  acetone,  bilirubin,  etc.,  will  be  considered  in  future 
chapters. 

Of  gases,  finally,  we  find  in  the  plasma  and  serum  small  amounts 
-of  nitrogen  and  oxygen,  which  are  present  simply  in  solution,  and 
somewhat  larger  amounts  of  carbon  dioxide,  which  in  part  at  least 
is  more  or  less  firmly  combined  with  albumins. 

The  Leucocytes. 

The  morphological  characteristics  and  general  chemical  composi- 
tion of  the  leucocytes  have  already  been  considered  (see  pages  303 
and  306).  At  this  place  I  wish  merely  to  draw  attention  to  one 
substance  which,  according  to  Lilienfeld,  is  found  in  special 
abundance  in  the  nuclei  of  these  bodies,  and  which  has  been  termed 
nucleohiston. 

Nucleohiston. — This  substance  was  first  isolated  by  Kossel  and 
Lilienfeld  from  the  thymus  gland  of  the  calf,  but  has  since  been 
obtained  from  the  leucocytes  of  the  lymph-glands,  as  also  from  the 
splenic  cells,  the  testicular  cells,  the  spermatozoa,  and  from  the 
epithelial  lining  of  the  small  intestine.  In  all  probability  it  repre- 
sents an  important  constituent  of  all  cellular  nuclei. 

Isolation. — Nucleohiston  is  most  conveniently  obtained  from  the 
leucocytes  of  the  thymus  gland.  To  this  end,  the  gland  is  carefully 
dissected  free  from  fat  and  all  larger  bloodvessels,  and  finely  hashed. 
This  mass  is  extracted  with  cold  water,  passed  through  muslin,  and 
centrifugalized.  The  aqueous  extract  is  further  filtered,  and  the 
nucleohiston  precipitated  by  the  careful  addition  of  dilute  acetic 
acid.  It  is  filtered  off,  dissolved  in  water  with  the  aid  of  a  small 
amount  of  a  dilute  solution  of  sodium  carbonate,  reprecipitated 
with  acetic  acid,  and  purified  by  a  repetition  of  this  process.  It  is 
then  washed  with  acetic  water,  extracted  with  alcohol  and  ether, 
and  finally  dried  at  a  temperature  of  from  110°  to  115°  C. 


CHEMICAL   EXAMINATION  OF   THE  BLOOD.  323 

Properties. — Thus  obtained,  nucleohiston  represents  a  snowy-white, 
fine  powder,  which  is  insoluble  in  benzol,  alcohol,  chloroform,  methyl 
alcohol,  ether,  and  acetic  acid,  but  is  soluble  in  water,  glacial  acetic 
acid,  concentrated  nitric  acid,  and  hydrochloric  acid,  in  solutions  of 
-odium  carbonate,  sodium  hydrate,  ammonia,  and,  when  freshly  pre- 
cipitated, also  in  solutions  of  sodium  chloride  and  magnesium  sul- 
phate, especially  in  the  presence  of  a  little  acetic  acid.  On  boiling 
with  water,  or  on  treating  with  baryta  water  or  dilute  hydrochloric 
acid,  nucleohiston  is  decomposed  into  a  nuclear  nuclein,  the  so-called 
leuconuclein,  and  an  albumose-like  substance,  liiston,  which  Kossel 
first  obtained  from  the  nuclei  of  the  red  corpuscles  of  the  goose.  It 
may  therefore  be  regarded  as  a  nucleo-albumin,  but  differs  from  most 
of  the  other  representatives  of  this  group  in  the  large  amount  of 
phosphorus — 3.025  percent. — which  it  contains.  The  histon  radicle 
possesses  marked  basic  properties,  and  readily  combines  with  acids. 
From  its  acid  solutions  it  is  precipitated  by  ammonia,  and  it  is 
insoluble  in  an  excess  of  the  reagent.  The  leuconuclein,  on  the 
other  hand,  has  markedly  acid  properties.  On  treating  with  an 
alcoholic  alkali  solution,  it  is  decomposed  into  an  albuminous  sub- 
stance and  a  nucleinic  acid,  which  is  termed  thymonueleinic  acid. 
On  further  decomposition  this  yields  an  acid  phosphoric  radicle, 
and  nucleinic  bases,  among  which  adenin  and  guanin  prevail.  On 
boiling  with  water  the  acid  radicle  is  transformed  into  so-called 
thyminic  acid,  which  on  treating  with  strong  sulphuric  acid  gives 
rise  to  thymin.  If,  on  the  other  hand,  the  nucleinic  acid  is 
treated  with  dilute  boiling  mineral  acids,  or  with  superheated  steam, 
adenin,  thymin,  and  another  basic  substance,  cytosin,  are  obtained, 
as  also  ammonia,  formic  acid,  phosphoric  acid,  and  lsevulinic  acid; 
the  presence  of  the  latter  suggests  that  in  the  nucleinic  acid  radicle 
a  carbohydrate  group  also  exists. 

The  nucleohiston,  as  such,  may  be  regarded  as  an  acid  salt,  as 
it  i-  capable  of  binding  a  certain  amount  of  calcium  and  sodium. 
Its  elementary  analysis  lias  given  the  following  results :  carbon,  48.46 ; 
hydrogen,  7;  nitrogen,  16.86;  phosphorus,  3.025  ;  sulphur,  0.701; 
and  oxygen,  23.95  per  cent. 

The  chemical  analysis  which  Lilienfeld  made  of  the  leucocytes 
of  the  thymus  gland,  and  which  probably  expresses  the  constitution 
of  all  leucocytes,  gave  the  following  results : 

Water 88.51  per  cent. 

ids 11.49  "  " 

Ubumine 1.76  ''  " 

Leuconuclein      68.78  "  " 

Burton 8.67  "  " 

Lecithin      7.51  "  " 

4.02  "  " 

Cholecterin      4.40  "  " 

Glycogen     0.80  "  " 

Nuclein  baset  ai  -ilver  salts 15.17  "  " 

1   .ill   phosphorus ."..01  "  " 

Total  nitrogen 15.03  "  " 


324  THE  BLOOD. 

Of  albumins  proper,  there  are  said  to  be  present  in  the  leucocytes 
the  so-called  cell-globulins,  of  which  Halliburton  recognizes  two — 
one  coagulating  at  50°  C,  the  other  at  73°  C. — and  one  of  which 
is  by  some  thought  to  be  identical  with  the  fibrin  ferment ;  then 
serum-albumin,  and  a  mucinous  body,  the  so-called  hyalin  sub- 
stance of  Rovida.  These  bodies,  however,  represent  only  a  very 
small  portion  of  the  solids  of  the  leucocytes,  as  is  seen  from  Lilien- 
felcl's  table,  and  it  is  doubtful  indeed  whether  their  true  chemical 
nature  has  been  sufficiently  established. 

The  Plaques. 

Of  the  chemical  composition  of  the  plaques  little  is  known. 
According  to  Lilienfeld,  they  contain  an  albuminous  substance  and 
a  nuclein  ;  for  on  treatment  with  artificial  gastric  juice  they  can  be 
differentiated  into  a  homogeneous  portion,  which  is  subsequently 
dissolved,  and  an  insoluble  granular  portion,  which  gives  the  vari- 
ous reactions  of  .the  nucleins,  and  may  be  shown  to  contain  phos- 
phorus. In  the  plaques  the  albumin  is  probably  combined  with  the 
nuclein  to  form  a  nucleo-albumin,  which  may  be  identical  with  the 
nucleohiston,  which  has  just  been  described.  According  to  Lilien- 
feld, indeed  the  plaques  must  be  regarded  as  nuclear  derivatives,  and 
he  has  accordingly  termed  them  the  nuclein  platelets  of  the  blood. 

The  Coagulation  of  the  Blood. 

According  to  modern  ideas,  the  coagulation  of  the  blood  is  ref- 
erable in  the  first  instance  to  the  decomposition  of  fibrinogen 
into  fibrin  and  fibrinoglobulin.  This  decomposition,  is  thought 
to  be  effected  through  the  activity  of  a  special  ferment,  the 
fibrin  ferment,  which  is  supposedly  contained  in  the  cellular  ele- 
ments of  the  blood  in  the  form  of  a  pro-enzyme,  and  is  set  free 
during  the  death  of  these  elements  as  a  calcium  compound  of 
the  pro-enzyme.  According  to  Pekelharing,  the  ferment  then  trans- 
fers its  calcium  to  the  fibrinogen,  which  is  thus  decomposed,  and 
is  itself  reconverted  into  the  pro-enzyme,  and  as  such  imme- 
diately combines  with  calcium,  which  is  again  transferred  to  an- 
other portion  of  fibrinogen,  and  so  on.  This  theory  is  in  all  like- 
lihood correct  in  its  general  features,  but  has  probably  not  yet 
assumed  its  ultimate  form.  That  other  substances,  besides  fibrin- 
ogen, a  small  amount  of  a  neutral  and  a  soluble  calcium  salt,  and 
the  fibrin  ferment,  need  not  be  present  to  effect  coagulation,  is  cer- 
tainly beyond  dispute.  But,  on  the  other  hand,  we  must  admit  that 
our  knowledge  of  the  fibrin  ferment  itself  and  the  mode  of  action 
of  the  calcium  salt  is  still  imperfect.  According  to  Halliburton,  the 
fibrin  ferment  is  a  cell-globulin  and  is  contained  as  such  in  the 
bodies  of  the  leucocytes.  Pekelharing,  on  the  other  hand,  regards 
the  ferment  as  a  calcium  compound  of  a  nucleo-albumin.    Lilienfeld, 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  325 

while  not  denying  the  existence  of  the  ferment,  regards  it  as  a  de- 
composition-product which  is  formed  during  the  coagulation  of  the 
blood  According  to  his  ideas,  it  cannot  very  well  be  concerned  in 
this  process,  and  he  assumes  that  the  coagulation  of  the  blood  is 
normally  referable  to  the  acid  radicle  of  the  nucleohiston,  which,  as 
we   have   seen,  is  present  in  the  nuclei  of  the  leucocytes  in  large 

amounts.  _    , 

The  leuconuclein,  however,  does  not  cause  the  coagulation  ot  the 
blood  at  once,  but  first  effects  the  decomposition  of  fibrinogen  with 
the  formation  of  thrombosin  and  an  albumose-like  body,  which  may 
be  a  derivative  of  Hammarsten's  fibrinoglobulin.  The  thrombosin 
is  soluble  in  dilute  alkalies.  Upon  the  addition  of  a  soluble  calcium 
salt,  however,  such  solutions  coagulate,  and  the  thrombosin  is  thus 
transformed  into  the  insoluble  fibrin.  Fibrin,  according  to  this 
theory,  is  thus  a  calcium  compound  of  thrombosin. 

The  active  principle    of   the  leuconuclein  is  unquestionably    its 
nucleinic  acid   radicle,  and,  as  a  matter  of  fact,  the  same  changes 
can  be  brought  about    by  using  this  directly.     This   explains  the 
observation  which  has  been  repeatedly  made,  that  the  coagulation 
of  albuminous  fluids  which  are  not  spontaneously  coagulable  can  also 
be  effected  by  the  addition  of  almost  any  cellular  formation,  such 
as  yeast  cells',  various  bacteria  arid  moulds,  spermatozoa  and  pro- 
tozoa, etc.—/.  e.,  bodies  which  all  contain  fairly  large  amounts  of 
nuclei ns.     It  is  to  be  noted,  moreover,  that  the  intensity  of  action 
of  these  various  substances  is  intimately  dependent  upon  the  amount 
of  nuclein  present,  and  we  accordingly  find  that  of  aH  the  cellular 
elements  of  the  body  the  spermatozoa  are  the  most  active.     While 
this  portion  of  Lilienfeld's  theory  is  practically  unassailable,  some 
doubt  has  been  raised  as  to  the  existence  of  his  soluble  thrombosin, 
and   Hammarsten  thus  states  that  the  thrombosin  is  no  decomposi- 
tion-product of  fibrinogen,  but  fibrinogen  itself  which  has  been  pre- 
cipitated   with  nucleinic  acid.     He  has  shown,  moreover,  that  the 
formation  of  fibrin  can  take  place  in  the  absence  of  calcium  salts, 
providing  that  a  sufficient  amount  of  the  fibrin  ferment  is  present. 
If  we  are  to  accept  Lilienfeld's  theory  then,  we   may  imagine  that 
through  the  influence  of  the  soluble  calcium  salt  the  nucleohiston  is 
decomposed,  and  that  the  leuconuclein  is  thus  enabled  to  exercise  its 
special   activity.     Future   researches,  however,  will  be  necessary  to 
aettle  this  question   definitely,  and  to  determine  whether  the  coagu- 
lation  of  the    blood    is    normally  referable  only    to   the  action  of 
nucleins,  and  whether  the  fibrin*  ferment  is  in  reality  a  product  of 
coagulation. 

The  question,  why  the  blood  does  not  coagulate  within  the  vessels 
of  the  body  where  fibrinogenic  material  is  available,  and  where  leuco- 
cytes no  doubt  undergo  degeneration,  has  been  variously  answered. 
On  the  one  hand,  it  is  stated  thai  the  integrity  of  the  endothelial 
lining  i-  hen- of  prime  importance,  ami  thai  coagulation  will  occur 
whenever  this  ie  impaired.     As  a  matter  of  fact,  we  find  that  coagu- 


326  THE  BLOOD. 

lation  takes  place  after  ligation  of  an  artery,  and  that  the  coagulum 
invariably  extends  as  far  as  the  next  collateral  vessel.  That  the 
nutrition  of  the  intima  is  here  seriously  interfered  with  cannot  be 
doubted.  Similarly  we  find  a  more  or  less  extensive  thrombosis 
in  atheromatous  vessels,  not  to  speak  of  the  process  of  clotting  in 
association  with  wounds.  In  such  cases  it  appears  that  owing  to  the 
lesion  of  the  endothelial  coat  an  aggregation  of  leucocytes  occurs 
in  the  affected  parts,  which  in  turn  results  in  the  death  and  disso- 
lution of  many  of  the  cells  at  these  places.  Contact  with  a  foreign 
substance,  and  as  such  diseased  or  dying  endothelial  cells  must  be 
viewed,  in  some  manner  brings  about  the  early  dissolution  of  the 
leucocytes,  and  we  find  accordingly  that  on  introducing  a  silk  thread 
into  the  bloodvessel  of  a  living  animal  coagulation  takes  place 
around  the  foreign  body.  Similarly,  it  may  be  observed  that  when 
blood  is  received  in  a  vessel,  the  walls  of  which  have  been  carefully 
lubricated  with  vaselin,  coagulation  is  greatly  delayed,  but  may  be 
brought  about  at  once  on  introducing  bits  of  foreign  material,  such 
as  dust  or  ashes  and  the  like. 

While  we  have  thus  seen  that  coagulation  will  occur  within  the 
living  body  whenever  foreign  material  is  present,  we  have  not  as  yet 
offered  an  explanation  for  the  non-occurrence  of  coagulation  when 
such  influences  are  not  at  work.  That  leucocytes  are  constantly 
broken  down  in  the  living  organism  cannot  be  questioned.  It  is 
possible,  however,  that  the  amount  of  nucleohiston,  or  fibrin  ferment, 
as  the  case  may  be,  which  is  thus  formed,  is  too  small  at  any  one 
time  to  exert  its  special  activity.  On  the  other  hand,  it  is  conceiv- 
able that  such  decomposition  may  take  place  within  certain  organs 
of  the  body,  such  as  the  spleen  and  the  liver,  and  that  the  nucle- 
histon,  which  is  here  set  free,  is  again  utilized  in  the  construction  of 
new  cells.  The  nucleohiston,  moreover,  contains  another  radicle, 
the  albumose-like  histon,  which  has  exactly  the  opposite  effect  upon 
coagulation.  For  whereas  the  injection  of  leuconuclein  into  the  cir- 
culation of  living  animals  rapidly  brings  about  the  death  of  the 
animal  from  thrombosis,  histon  injections  render  the  blood  refractory 
to  coagulation.  Such  plasma  is  termed  histon-plasma,  and  it  is  to 
be  noted  that  this  coagulation  can  later  be  brought  about  only  through 
the  addition  of  the  leuconuclein  or  nucleinic  acid.  It  is  thus  possi- 
ble that  during  the  dissolution  of  the  leucocytes  in  the  living  animal 
the  leuconuclein  is  in  some  manner  prevented  from  exercising  its 
special  activity,  while  the  histon  further  prevents  coagulation  in 
itself.  The  primary  factor,  however,  upon  which  the  occurrence  or 
non-occurrence  of  coagulation  in  the  living  body  probably  depends, 
is  the  extent  to  which  the  leucocytes  are  undergoing  decomposition. 

Rapidity  of  Coagulation. — The  rapidity  with  which  coagu- 
lation of  the  blood  occurs  after  being  shed  varies  with  different 
animals,  with  the  districts  from  which  the  blood  is  taken,  etc. 
In  birds  it  thus  occurs  after  one  and  a  half  minutes ;  in  man 
after  from  three  to  four   minutes ;  while    in  cold-blooded   animals 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  327 

it  begins  onlv  after  a  quarter  of  an  hour.  In  the  horse,  in  which 
coagulation  is  Likewise  delayed,  the  corpuscles  of  the  blood  have  time 
to  settle  and  form  two  distinct  layers— the  red  at  the  bottom  and  the 
white  on  top,  where  they  appear  as  a  grayish-white  zone,  and  con- 
stitute the  so-called  crusta  phhgistica  or  infiammatoria.  Above  this 
we  then  see  the  plasma,  which  has  undergone  coagulation,  and  on 
top  of  it  the  serum  that  has  been  squeezed  out  from  the  clot.  The 
same  phenomenon  may  be  observed  in  human  blood  when  cooled  to 
about  0°  C,  and  from  the  extent  of  the  crusta  phlogistica  in  such 
blood  the  older  physicians  were  wont  to  draw  prognostic  conclusions 
as  to  the  course  of  the  disease. 

The  rapidity  of  coagulation  can  be  artificially  increased  and 
diminished.  Bv  beating  the  blood,  by  increasing  its  temperature 
a  little  beyond  that  of  the  body,  and  by  diluting  with  water  it  is 
increased  ;"  while  exposure  to  a  low  temperature,  the  presence  of 
much  carbon  dioxide  in  the  blood,  the  careful  lubrication  of  the 
vessel  with  vaselin  or  similar  unguents,  cause  a  retardation  of  coagu- 
lation. Its  prevention  finally  may  be  brought  about  through  influ- 
ences already  mentioned,  viz.,  by  salting  with  the  neutral  salts, 
following  the  previous  injection  into  the  body  of  albumoses  or  of 
histon,  of  diastatic  ferments,  of  extracts  of  the  mouth-parts  of  the 
leech,  after  elimination  of  the  intestinal  bloodvessels  by  ligation,  etc. 

The  Red  Corpuscles. 

The  red  corpuscles  of  the  blood,  as  has  been  mentioned,  owe  their 
color  to  the  presence  of  haemoglobin  or  its  oxygen  compound,  oxy- 
hemoglobin. This  may  be  extracted  by  diluting  with  water,  by 
alternate  freezing  and  thawing,  by  shaking  with  ether,  chloroform, 
etc.  The  blood  is  thereby  rendered  lake-colored,  and  on  microscopi- 
cal examination  it  will  be  observed  that  instead  of  the  original  cor- 
puscles,  BO-called  blood-shadows  are  now  found.  These  are  colorless 
ring-like  bodies,  and  constitute  the  stroma  of  the  red  corpuscles. 
In  the  circulating  blood  the  dissolution  of  the  haemoglobin  is  pre- 
vented by  the  presence  of  large  amounts  of  sodium  chloride.  Such 
blood  is  -aid  to  be  hyperi&otonic — i.  e.,  it  contains  more  sodium 
chloride  than  is  necessary  to  prevent  the  dissolution  of  the  coloring- 
matter  from  its  corpuscles.  Within  the  corpuscles  the  haemoglobin 
i-,  however,  supposedly  not  present  in  the  free  state,  but  in  com- 
bination with  some  other  substance,  such  as  lecithin;  and  Hoppe-: 
Sevier  accordingly  distinguishes  between  the  so-called  arterin  and 
phlebin,  which  represents  the  lecithin  compound  of  oxyhemoglobin 
and  haemoglobin  respectively. 

A-  has  been  mentioned,  the  red  corpuscles  represent  nearly  one- 
half  of  the  liquid  blood.  They  contain  aboul  o7.7  per  cent,  of 
water  and  10.5  per  cent,  of  oxyhemoglobin,  while  tin'  constituents 
of  the  stroma  inclusive  of  mineral  salts  amounl  only  to  about  1.9  per 
cent.     A.mone  these  constituents  Halliburton's  cell-globulin  is  said 


328  THE  BLOOD. 

to  be  most  abundant ;  in  addition  we  find  traces  of  lecithin,  choles- 
terin,  and  nucleo-albumin,  while  serum-albumin  and  albumoses  are 
apparently  absent.  In  the  nucleated  red  corpuscles  of  birds  we 
further  meet  with  the  integral  constituents  of  the  nuclei,  among 
which  Lilienfeld's  nucleohiston  probably  always  prevails.  Its  basic 
constituent,  histon,  was  first  discovered  by  Kossel  in  the  red  cor- 
puscles of  the  goose. 

An  idea  of  the  mineral  constituents  of  the  red  corpuscles,  viz., 
their  stroma,  may  be  had  from  the  following  table,  which  is  taken 
from  C.  Schmidt,  and  calculated  for  100  parts  of  the  moist  cor- 
puscles. 

Potassium  chloride 3.68 

Sodium  chloride traces. 

Potassium  sulphate      0.13 

Potassium  phosjmate 2.34 

Sodium  phosphate 0.63 

Calcium  phosphate       0.09 

Magnesium  phosphate 0.06 

The  iron  of  the  haemoglobin  is  not  included  in  this  table ;  in  man 
it  varies  between  0.506  and  0.537  pro  mille.  In  addition  traces  of 
copper  are  not  infrequently  met  with,  and,  as  will  be  seen  later,  this 
element  in  some  of  the  invertebrate  animals  apparently  takes  the 
place  of  iron  in  the  coloring-matter  of  the  blood. 

Isolation  of  the  Red  Corpuscles  of  the  Blood. — To  isolate  the 
red  corpuscles,  the  blood  is  defibrinated  by  beating,  diluted  with  ten 
times  its  volume  of  a  1  per  cent,  solution  of  sodium  chloride,  and 
passed  through  a  muslin  filter.  On  subsequent  centrifugation  and 
repeated  washing  with  the  salt  solution,  they  are  then  freed  from  the 
serum,  and  may  now  be  collected  on  a  paper  filter,  after  the  previous 
addition  of  a  large  amount  of  alcohol.  Any  fats,  lecithins,  or 
cholesterins  that  may  be  present  are  extracted  with  warm  alcohol 
and  ether,  when  the  corpuscles  can  be  dried  and  weighed.  To 
isolate  the  stromata,  the  mass  of  corpuscles,  when  freed  from  serum, 
is  shaken  with  five  or  six  times  its  volume  of  water  and  a  small 
amount  of  ether.  The  mixture  is  then  centrifugalized,  and  is  thus 
separated  from  the  leucocytes.  The  stromata  are  now  precipitated 
by  adding  a  few  drops  of  a  1  per  cent,  solution  of  acid  sodium 
phosphate,  until  the  liquid  has  almost  assumed  the  consistence  of 
the  original  blood.  They  are  then  collected  on  a  filter,  quickly 
washed  with  water,  and  may  now  be  dissolved  in  a  5  per  cent,  solu- 
tion of  magnesium  sulphate. 

Haemoglobin  and  Its  Derivatives. 

Haemoglobin. — The  hsemoglobin  is  the  coloring-matter  of  the 
red  corpuscles,  and  is  present  in  these  in  combination  with  an- 
other body,  which  may  be  a  lecithin,  as  the  phlebin  of  Hoppe- 
Seyler,  while  the  corresponding  compound  of  oxyhemoglobin  is 
termed    arterin.     Of    the    nature   of    these    compounds,    however, 


CHEMICAL  EXAMINATION  OF  THE  BLOOD. 


329 


but  little  is  known,  and   it  has  not  even  been  definitely  ascertained 
that  the  pairling  of  the.  coloring-matter  is  really  a  lecithin. 

Hemoglobin  or  its  oxy-compound  occurs  widely  distributed  in 
the  animal  world,  and  is  found  not  only  in  the  vertebrates,  but  also 
in  many  of  the  invertebrates.  But  while  in  the  former  it  is  con- 
tained in  definite  cellular  elements  of  the  blood  it  may  also  occur  as 
such  among  certain  invertebrate  animals.  Closely  related  to  it  is 
the  so-called  oxyhcemocyanin,  which  is  found  in  certain  arthropods 
and  molluscs,  and  in  which  the  iron  is  apparently  replaced  by 
copper  Then  again  we  find  among  invertebrate  animals  various 
violet  and  purplish-red  pigments,  the  so-called  floridins,  which  are 
likewise  to  be  classed  with  haemoglobin,  and  as  we  have  previously 
seen  a  genetic  relationship  apparently  also  exists  between  haemo- 
globin and  the  chlorophyl  of  plants.  These  various  pigments  are 
collectively  spoken  of  as  respiratory  pigments,  as  they  are  intimately 
concerned"  in  the  transportation  of  the  oxygen  of  the  air  to  the 
various  tissues  of  the  body,  and  in  the  removal  of  the  carbon 
dioxide  which  results  as  a  product  of  cellular  metabolism. 

Outside  of  the  blood  haemoglobin  is  found  also  in  striped  and  un- 
striped  muscle-tissues,  and  under  pathological  conditions  it  may 
appear  in  the  urine  as  such.  Different  haemoglobins  apparently 
exist  It  is  hence  impossible  to  give  a  definite  formula  which  ex- 
presses the  constitution  of  all.  An  idea  of  their  quantitative  ele- 
mentary composition  may  be  had  from  the  accompanying  table  : 

Carbon.  Hydrogen.  Nitrogen.  Oxygen.  Sulphur.  Phosphorus.    Iron. 

Horse 54.87         6.97         17.31         19.730  0.650           .    .          0.470 

Sof   .....  54.57         7.22         16.38         20.930  0.568           .   .          0.336 

P& 54.71         7.38         17.43         19.602  0.479           .    .          0.399 

Guinea-pi"     .    .    •    -54.12        7.36         16.78         20.680  0.580           .    .          0.480 

Sau"rrel  .    .    .54.09         7.39         16.09         21.440       0.400  .   .  0.590 

hquirrei  o.  0  540  Q  770        0  430 

aSkea' : : : :  :ili?    f.lS    &3    22.500  0.857    0.197   0.335 

The  size  of  the  hamoglobin  molecule  is,  like  that  of  all  albu- 
minous substances,  very  Targe.  For  that  of  dog's  blood  Hiifner 
obtained  the  figure  14,129,  which  would  correspond  to  the  formula 
C  II  X,  FeS3Owl.  It  thus  contains  three  atoms  of  sulphur  for 
one  atom  of  iron,  while  the  haemoglobin  of  the  horse  and  pig  has 
only  two  atoms  of  sulphur  for  one  atom  of  iron.  Of  interest 
further  is  the  presence  of  phosphorus  in  the  hasmoglobin  of  the 
goose  and  chicken.  Whether  this  forms  an  integral  component  of 
the  bsemoglobiii  molecule,  however,  is  questionable,  and  it  is  quite 
possible  thai  its  presence  is  owing  to  a  contamination  of  the  coloring- 
matter  with  nucleinic  acid  derived  from  the  nuclei  of  the  red 
corpuscles. 

Structurally,  haemoglobin  must  be  regarded  as  a  proteid,  viz.,  as  a 
compound  of  an  albuminous  radicle  with  another  complex  organic 
radicle.  This  other  radicle  U  here  an  iron-containing  pigment, 
which  may  be  separated  from  its  albuminous  pairling,  and  is  termed 


330  THE  BLOOD. 

hcemochromogen  (see  below),  while  the  albuminous  substance  is 
known  as  globin.  These  two  substances  are  apparently  united  in 
the  haemoglobin  molecule,  through  an  additional  radicle,  which  is  as 
yet  unknown. 

Globin,  is  closely  related  to  histon,  and,  like  it,  presents  the  fol- 
lowing characteristic  reactions  :  it  is  precipitated  by  ammonia  from 
its  solutions  in  dilute  hydrochloric  acid,  and  is  insoluble  in  an  excess 
of  the  reagent.  With  concentrated  nitric  acid  it  is  thrown  down  in 
the  cold,  but  not  from  its  heated  solutions.  Under  certain  condi- 
tions it  can  be  coagulated  on  boiling,  but,  unlike  the  other  coagula- 
ble  albumins,  its  coagulate  is  readily  soluble  in  acids.  It  contains 
54.97  per  cent,  of  carbon,  7.2  per  cent,  of  hydrogen,  16.89  per  cent, 
of  nitrogen,  and  0.42  per  cent,  of  sulphur.  The  amount  of  globin 
which  can  be  obtained  from  the  haemoglobin  molecule  is  quite  large, 
and  according  to  Schultz  amounts  to  86.5  per  cent.,  while  4.2  per 
cent,  only  is  represented  by  the  pigment  itself. 

Isolation. — A  solution  of  oxyhemoglobin  in  water,  prepared  at 
a  temperature  of  40°  C,  is  treated  with  dilute  hydrochloric  acid 
until  the  red  color  changes  to  brown.  This  mixture  is  extracted 
with  80  per  cent,  alcohol  (one-fifth  volume)  and  ether  (one-half 
volume)  until  the  ether  takes  up  no  more  coloring-matter.  The 
resulting  aqueous-alcoholic  solution  is  precipitated  with  ammonia, 
filtered,  and  the  precipitate  dissolved  in  very  dilute  acetic  acid.  On 
filtering,  the  globin  is  again  precipitated  with  ammonia  and  col- 
lected on  a  silk  filter.  After  washing  with  absolute  alcohol,  then 
with  water,  again  with  alcohol,  and  finally  with  ether,  the  substance 
is  dried  first  in  the  air  and  then  at  a  temperature  of  100°  C.  The 
resulting  material  constitutes  pure  globin,  as  a  yellowish  loose 
powder,  which  is  not  especially  hygroscopic. 

Haemochromogen. — The  isolation  of  haemochromogen  is  rather 
difficult,  owing  to  the  avidity  with  which  it  combines  with  oxygen  to 
form  hcematin  in  alkaline  solution.  Hoppe-Seyler,  however,  suc- 
ceeded in  obtaining  the  substance  in  crystalline  form,  by  heating 
haemoglobin  with  sodium  hydrate  solution  in  an  atmosphere  of  hy- 
drogen. In  acid  solutions  haemochromogen  gradually  loses  its  iron 
and  is  converted  into  hcematoporphyrin.  In  alkaline  solution  it 
presents  a  beautiful  cherry-red  color,  and  on  spectroscopic  examina- 
tion gives  two  bands  of  absorption.  One  of  these  is  very  intense, 
and  located  between  D  and  E,  nearer  D,  while  the  other  is  not  so 
dark,  but  wider,  and  found  about  E  and  extending  beyond  b.  To 
demonstrate  the  spectrum  of  hsemochromogen,  bloody  fluid  is  mixed 
with  a  solution  of  sodium  hydrate,  when  the  resulting  haematin  is 
reduced  with  ammonium  sulphide,  or  Stokes  reagent,  viz.,  an  am- 
moniacal  solution  of  ferrous  tartrate,  or  stannous  chloride. 

The  haemochromogen  radicle,  as  has  been  stated,  represents  the 
pigmented  group  of  the  haemoglobin  molecule,  and  to  its  presence 
the  power  of  haemoglobin  to  combine  with  oxygen,  carbon  dioxide, 
and  other  gases,  is  unquestionably  due. 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  331 

Hemoglobin  itself  can  be  obtained  in  crystalline  form,  and  is 
characterized  by  the  great  resistance  which  it  offers  to  putrefaction 
and  tryptic  decomposition.  It  is  stated  that  even  after  the  lapse  of 
years  decomposed  blood  contains  its  haemoglobin  as  such,  and  that 
on  shaking  with  air  it  may  again  be  transformed  into  pure  oxy- 
hemoglobin. Its  solutions  present  a  beautiful  purplish-red  color, 
and  on  spectroscopic  examination  gives  rise  to  a  single  band  of 
absorption,  which  lies  between  D  and  E  and  extends  slightly  to  the 
left  beyond  D.  This  is  most  conveniently  shown  by  taking  a  solu- 
tion of  oxyhemoglobin,  and  reducing  this  with  ammonium  sulphide 
or  Stokes'  fluid,  as  directed  above. 

Most  important  is  the  avidity  with  which  haemoglobin  combines 
with  various  gases,  and  upon  this  characteristic  indeed  its  chief 
function,  as  a  respiratory  pigment,  is  based.  This  property,  as  has 
been  stated,  is  referable  to  the  chromogen  radicle,  and  more  partic- 
ularly to  the  iron,  which  it  contains.  Every  atom  of  this  is  capable 
of  combining  with  one  molecule  of  oxygen  or  of  carbon  dioxide, 
and  in  this  form  largely  the  oxygen  of  the  air  is  carried  to  the  vari- 
ous tissues  of  the  body,  and  the  carbon  dioxide  removed.  In  the 
circulating  blood  we  accordingly  find  only  relatively  small  amounts 
of  free  haemoglobin,  and  in  arterial  blood  its  oxy-compound  is 
almost  exclusively  encountered. 

The  amount  of  haemoglobin  which  is  contained  in  human  blood, 
either  as  such  or  in  combination  with  oxygen  or  carbon  dioxide,  is 
about  14  per  cent.,  but  subject  to  certain  variations,  even  in  health, 
while  in  disease  still  greater  deviations  from  the  average  normal 
amount  are  observed.  A  great  diminution  may  here  occur,  and  is 
most  marked  in  chlorosis  and  pernicious  anaemia,  in  which  the  per- 
centage may  fall  as  low  as  2.35. 

As  the  isolation  of  the  haemoglobin  from  the  blood  resolves 
itself  into  the  isolation  of  its  oxy-compound,  this  will  be  considered 
together  with   its  quantitative  estimation  under  that  heading. 

Oxyhaemoglobin. — Oxyhaemoglobin  is  the  oxy-compound  of  haemo- 
globin, and  differs  from  its  mother-substance  in  containing  two 
at'. ni~  more  of  oxygen,  which  are  bound  to  the  one  atom  of  iron, 
than  are  present  in  the  haemochromogen  radicle.  In  this  manner, 
however,  the  haemochromogen  is  converted  into  haematin,  and  we 
may  therefore  say  that  oxyhaemoglobin,  in  contradistinction  to 
haemoglobin,  consists  of  the  globin  radicle  united  by  some  unknown 
group  to  a  haematin  radicle  (see  also  page  333). 

Haematin. — In  accordance  with  the  above  considerations,  we 
find  that  on  decomposition  of  oxyhaemoglobin  haematin  is  obtained 
i r i - 1 * -; i « I  of  haemochromogen.  This  latter,  indeed,  is  at  once  trans- 
formed into  haematin  on  exposure  to  oxygen,  and,  as  we  have  seen, 
the  haematin  is  correspondingly  reconverted  into  haemochromogen  by 
treating  with  reducing  agents.  The  decomposition  of  oxynaemo- 
globin  with  th<'  formation  of*  haematin  can  be  readily  effected  by 
Beating  its  solutions  to  a  temperature  of  80"  C,  by  treating  with 


332  THE  BLOOD. 

dilute  mineral  acids,  or  with  stronger  solutions  of  the  alkaline 
hydrates,  as  also  by  peptic  or  tryptic  digestion.  To  obtain  the  sub- 
stance in  a  pure  state,  however,  it  is  best  to  start  with  its  hydro- 
chlorate  anhydride,  hcemin,  which  can  be  readily  obtained  in  crystal- 
line form.  To  this  end,  oxyhemoglobin  is  treated  with  a  trace  of 
sodium  chloride  and  dissolved  in  glacial  acetic  acid.  On  heating, 
the  hsemin  is  precipitated  in  the  form  of  very  characteristic,  drawn- 
out,  rhombic  platelets,  which  are  insoluble  in  water,  alcohol,  and 
ether,  with  difficulty  so  in  glacial  acetic  acid  and  in  dilute  mineral 
acids,  but  are  readily  soluble  in  dilute  alkaline  solutions  and  acid 
alcohol.  The  crystals  are  collected  on  a  filter,  washed  with  alcohol 
and  ether,  and  are  thus  obtained  in  pure  form.  To  prepare  the 
hsematin,  the  hsemin  is  dissolved  in  a  dilute  solution  of  sodium 
hydrate  and  supersaturated  with  dilute  hydrochloric  acid.  The  sub- 
stance is  thus  precipitated  in  the  form  of  brownish  flakes,  which  are 
washed  free  from  chlorides  and  dried  at  120°  C.  Hsematin,  in  con- 
tradistinction to  oxyhemoglobin  and  hsemin,  is  non-crystallizable. 
Its  solubility  is  essentially  the  same  as  that  of  hsemin.  In  acid  solu- 
tion both  hsematin  and  hsemin  show  a  well-defined  spectral  band 
"between  C  and  D.  Between  D  and  F  a  second  band  is  seen,  which 
is  much  wider  but  less  sharply  defined  than  the  first.  By  diluting 
the  solution  this  band  may  be  resolved  into  three  bands,  of  which 
one  is  located  between  b  and  F,  near  F ;  another,  between  D  and  E, 
nearer  E ;  and  a  third  faint  band  between  D  and  E,  nearer  D.  As 
a  rule,  however,  only  two  bands  are  seen.  The  alkaline  solutions  of 
hsematin  are  distinctly  dichrotic  and  give  only  one  band  of  absorp- 
tion, the  greater  portion  of  which  lies  between  C  and  D,  and  extends 
slightly  beyond  D. 

On  careful  oxidation  hsematin  may  give  rise  to  an  iron-containing 
body  and  dibasic  hsematinic  acid,  C8H10O5,  which  can  be  further 
transformed  into  the  tribasic  acid,  C8H10O6.  Of  the  nature  of  these 
substances,  however,  but  little  is  known.  Of  greater  interest  is  the 
fact  that  on  treatment  with  concentrated  sulphuric  acid,  with  hydro- 
chloric acid,  or  with  glacial  acetic  acid  and  hydrobroinic  acid, 
hsematin  as  well  as  hsemin  is  freed  from  iron,  and  on  the  subsequent 
addition  of  water  is  transformed  into  hsematoporphyrin,  C32H36N406, 
which  is  isomeric  with  bilirubin  (see  below). 

Hsematin  is  thus  a  decomposition-product  of  oxyhsemoglobin,  and 
is  not  found  as  such  in  the  circulating  blood.  It  is  said  to  occur  in 
the  urine,  however,  in  cases .  of  poisoning  with  arsenious  hydride. 
In  the  stools  it  is  found  after  hemorrhages  into  the  stomach  or  the 
upper  portion  of  the  small  intestine,  and  also  after  the  ingestion  of 
large  amounts  of  red  meats.  In  such  cases,  of  course,  its  origin  is 
referable  to  the  decomposition  of  hsemoglobin  through  the  agency  of 
the  gastric  and  pancreatic  juices. 

Isolation  of  Oxyhsemoglobin. — Oxyhsemoglobin  in  crystalline  form 
is  best  obtained  from  the  red  corpuscles  of  the  horse  or  dog, 
according   to   the  following  method :    the   blood   is   first  rendered 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  333 

uncoacmlable  by  treating  with  ammonium  oxalate  (0.06-0.1  per 
cent.),°and  is  then  centrifugalized  to  effect  the  separation  of  the  cor- 
puscles. This  mass,  when  freed  from  plasma  by  siphonage,  is  treated 
with  twice  its  volume  of  water  and  placed  on  ice.  The  resulting 
fluid  is  now  mixed  with  an  equal  volume  of  a  saturated  solution  oi 
ammonium  sulphate  that  has  likewise  been  cooled  to  a  low  tempera- 
ture. This  mixture  is  also  placed  on  ice  until  the  precipitate  of 
globulins,  which  is  referable  to  remaining  plasma,  has  settled.  On 
filtering  in  the  refrigerator  a  perfectly  clear  dark-red  filtrate  is 
obtained,  which  contains  the  greater  portion  of  the  coloring-matter. 
If  now  the  solution  is  brought  to  the  temperature  of  the  room, 
the  separation  of  crystalline  oxyhemoglobin  begins,  and  may  be 
hastened,  if  necessary,  by  the  further  addition  of  a  small  amount 
of  the  ammonium  sulphate  solution.  After  a  few  days  the  process 
is  completed,  when  the  crystals  are  filtered  off  through  a  Biichner 
filter  bv  the  aid  of  a  suction  pump,  and  are  partially  freed  from  the 
mother-liquor  bv  pressing  between  filter-paper.  The  substance  is 
then  purified  by  recrystallization.  To  this  end,  the  crystalline 
mass  is  dissolved  in  water,  reprecipitated  by  the  addition  of  an  equal 
volume  of  the  ammonium  sulphate  solution,  and  so  on,  until  the 
required  degree  of  purity  has  been  attained.  Adhering  ammonium 
sulphate  is  finally  removed  mechanically.  To  preserve  the  sub- 
stance, it  is  dried  in  a  vacuum  or  at  a  low  temperature  over  sulphuric 
acid,  and  is  then  quite  stable. 

The  ease  with  which  oxyhemoglobin  can  be  brought  to  crystal- 
lization differs  with  different  animals.  In  guinea-pigs,  squirrels,  and 
rats  it  is  most  pronounced ;  and  it  is  here  only  necessary  to  mix  a 
few  drops  of  the  blood  with  an  equal  amount  of  water,  when  the 
process  may  be  directly  observed  with  the  microscope.  Human 
blood,  as  also  that  of  the  pig  and  the  ox,  is  much  more  refractory, 
and  is  not  well  adapted  for  the  preparation  of  the  pigment  in  its 
crystalline  state. 

The  form  of  the  crystals  varies  in  different  animals.  We  thus 
find  hexagonal  platelets  in  the  squirrel,  rhombic  tetrahedra  in 
the  guinea-pig  and  various  birds,  rhombic  needles  in  man,  etc. 

In"  its  chemical  behavior  oxyhemoglobin  manifests  its  albuminous 
nature.  It  is  thus  coagulated  on  boiling,  and  on  treating  with 
absolute  alcohol  and  most  of  the  salts  of  the  heavy  metals.  It  is  to 
be  noted,  however,  that  the  process  of  coagulation  is  associated  with 
the  decomposition  of  the  compound  into  hematin  and  globin,  which 
latter  is  precipitated  in  its  coagulated  state.  From  its  solutions 
the  substance  IS  thrown  down  by  half-saturation  with  ammonium 
Sulphate.  On  reduction  with  ammonium  sulphide,  with  Stokes' 
reagent,  or  during  the  process  of  putrefaction,  it  is  transformed 
into  haemoglobin.  Other  reducing  agents,  such  as  sodium  hydro- 
Bulphite,  give  rise  to  the  formation  of  so-called  pseudohaemoglobin, 
which  apparently  Btanda  midway  between  oxyhemoglobin  and 
hemoglobin   in    containing  less  oxygen  than  the  former,  but  more 


334  THE  BLOOD. 

than  the  latter.  Its  spectrum,  however,  is  the  same  as  that  of  the 
completely  reduced  haemoglobin. 

In  sufficiently  dilute  solution  oxyhemoglobin  shows  two  bands 
of  absorption  between  D  and  E.  The  one  to  the  left,  which  is  not 
so  wide  as  the  other,  but  darker  and  more  sharply  denned,  borders 
on  D,  while  the  second  lies  at  E. 

The  Quantitative  Estimation  of  Oxyhemoglobin. — This  is  best 
accomplished  by  Hoppe-Seyler's  method,  which  is  based  upon 
the  comparison  of  a  given  amount  of  diluted  blood  with  a  stand- 
ard solution  of  crystallized  oxyhemoglobin.  This  solution  is 
prepared  by  dissolving  2  grammes  of  the  pure  coloring-matter  in 
50  c.c.  of  distilled  water.  The  oxyhemoglobin  is  then  transformed 
into  carbon  monoxide  hemoglobin  by  passing  a  current  of  the  gas 
through  the  solution  to  saturation.  It  is  then  stored  in  drawn- 
out  and  sealed  glass  tubes,  such  that  each  tube  contains  about 
6  c.c.  The  contents  of  each  tube,  when  diluted  with  ten  times  its 
volume  of  water,  will  then  represent  a  0.2  per  cent,  solution  of  the 
oxyhemoglobin. 

A  carefully  measured  or  weighed  amount  of  blood,  not  exceed- 
ing 0.5  c.c,  is  now  diluted  with  water  that  has  been  saturated  with 
carbon  monoxide  to  exactly  5  c.c.  A  small  drop  of  a  very  dilute 
solution  of  sodium  hydrate  is  added  if  necessary  to  remove  any 
turbidity  that  may  exist.  This  solution  is  further  saturated  with  car- 
bon monoxide,  and  freed  from  fibrin  by  filtration.  The  filtrate  should 
measure  exactly  4  c.c.  The  comparison  of  the  two  solutions,  and 
the  further  dilution  of  the  blood  with  carbon  monoxide  water  then 
takes  place  in  the  so-called  double  pipette  of  Hoppe-Seyler.  The 
color  of  the  two  solutions  is  here  equalized,  and  the  amount  of 
hemoglobin  present  in  the  specimen  of  blood  calculated  from  the 
degree  of  dilution. 

Example. — Suppose  that  we  started  with  0.5  gramme  of  blood,  and 
that  the  standard  solution  contained  0.002  gramme  of  oxyhemoglo- 
bin in  the  cubic  centimeter.  The  4  c.c.  of  the  diluted  and  filtered  blood 
are  further  diluted  in  the  pipette  to  22  c.c,  which  corresponds  to  a 
total  solution  of  27.5  c.c.  for  the  total  5  c.c.  of  the  first  dilution.  In 
these  27.5  c.c,  which  represent  the  original  0.5  gramme  of  blood, 
there  will  consequently  be  27.5  X  0.002  =  0.0550  gramme  of 
hemoglobin.     The  percentage  will  accordingly  be  11  per  cent. 

In  the  clinical  laboratory  other  forms  of  apparatus  are  in  use, 
such  as  the  hosmometer  of  Fleischl  and  the  hcemoglobinometer  of 
G-owers.  In  the  first  the  color  of  the  diluted  blood  is  compared 
with  that  of  a  glass  wedge  that  has  been  stained  with  the  golden 
purple  of  Cassius.  In  the  second  a  standard  solution  of  carmin 
and  picric  acid  is  employed.  But  as  these  colors  do  not  represent 
the  exact  shade  of  oxyhemoglobin,  the  results  must  of  necessity  be 
less  accurate.  For  clinical  purposes,  however,  these  methods  are 
sufficiently  exact. 

The  spectro-photometric   determination  of   the   blood    coloring- 


CHEMICAL  EXAMINATION  OF  THE  BLOOD.  335 

mutter  is  not  described  at  this  place.  It  is  undoubtedly  the  most 
exact,  but  necessitates  the  use  of  costly  apparatus,  which  will  be 
found  in  only  few  laboratories. 

It  has  been  pointed  out  that  haemoglobin  is  characterized  by  the 
readiness  with  which  it  combines  with  certain  gases,  and  we  have 
just  considered  the  most  important  of  these  compounds.  Other 
compounds  of  this  character  are  carbon  dioxide  haemoglobin,  carbon 
monoxide  haemoglobin,  and  nitric  oxide  haemoglobin. 

Carbon  Dioxide  Haemoglobin. — Three  different  forms  are  said  to 
exist,  which  have  been  respectively  termed  a,  ft,  and  y  earbohcemo- 
globin,  but  they  are  comparatively  little  known.  According  to 
Bohr,  the  carbon  dioxide  in  these  compounds  is  united  with  the 
albuminous  radicle  of  the  haemoglobin,  while  the  oxygen  of  oxy- 
haemoglobin  is  combined  with  the  pigmented  group.  He  accordingly 
finds  that  when  a  solution  of  haemoglobin  is  shaken  with  a  mixture 
of  oxygen  and  carbon  dioxide  both  of  these  gases  are  taken  up  inde- 
pendently of  each  other. 

Carbon  Monoxide  Haemoglobin. — This  compound  results  from 
the  union  of  one  molecule  of  haemoglobin  with  one  molecule  of  carbon 
monoxide,  and  is  characterized  by  its  greater  stability  as  compared 
with  oxvhaemoglobin.  The  carbon  monoxide  is  in  this  case  united 
with  the  pigmented  radicle  of  the  haemoglobin,  and  may  be  split  off 
in  this  combination  as  carbon  monoxide  luemochromogen.  Like  the 
native  luemochromogen,  the  carbon  monoxide  compound  can  be 
obtained  in  crystalline  form,  and  on  exposure  to  the  air  is  likewise 
transformed  into  haematin.  Under  the  same  conditions  the  haemo- 
globin  compound  is  gradually  reconverted  into  oxyhemoglobin. 

Blood  containing  carbon  monoxide  haemoglobin  is  characterized 
by  its  cherry-rod  color,  its  resistance  to  putrefactive  changes  in  the 
absence  of  oxygen,  and  by  its  spectrum.  This  is  similar  to  that  of 
oxvhaemoglobin,  but  its  two  bands  of  absorption,  between  D  and  E, 
are  placed  rather  nearer  the  violet  end  of  the  spectrum.  Unlike  the 
spectrum  of  oxyhemoglobin,  however,  that  of  the  carbon  monoxide 
compound  is  not  changed  to  the  haemoglobin  spectrum  on  treating 
with  reducing  agents.  Should  oxyhemoglobin  be  simultaneously 
present,  a  mixed  spectrum  of  the  two  substances  is  obtained. 

Such  blood,  moreover,  when  treated  with  double  its  volume  of  a 
solution  of  sodium  hydrate  (sp.  gr.  1.3),  is  not  changed  to  a  dirty 
brownish  mass,  with  a  tint  of  green,  as  with  normal  blood,  but 
presents  a  beautiful  red  color,  which  changes  to  brown  only  on 
standing. 

In  it-  crystalline  state  carbon  monoxide  haemoglobin  may  be 
obtained  by  saturating  a  sufficiently  concentrated  solution  with  car- 
l.on  monoxide  and  cooling  the  mixture  to  0°  C, when  one-fourth  of 
it-  volume  of  cooled  alcohol  i-  added.  On  standing  in  the  refrigera- 
tor the  substance  separates  out  in  the  form  of  bluish-red  crystals, 
which  are  isomorphous  with  those  of  oxyhemoglobin,  but  much 
more  stable. 


336  THE  BLOOD. 

Nitric  Oxide  Haemoglobin. — This  compound  is  more  stable  even 
than  carbon  monoxide  haemoglobin,  and,  like  this,  may  be  obtained 
in  crystalline  form.  Its  spectrum  is  similar  to  that  of  carbon  mon- 
oxide haemoglobin.  The  bands,  however,  are  less  sharply  defined 
and  paler  than  those  of  that  compound,  and,  like  these,  do  not  dis- 
appear on  the  addition  of  a  reducing  agent.  The  substance  is  met 
with  in  poisoning  with  the  gas  in  question. 

Methaemoglobin. — Methaemoglobin  is  a  pigment  which  normally 
does  not  occur  in  the  blood,  but  is  found  after  the  ingestion  of  large 
amounts  of  potassium  chlorate,  antifebrin,  potassium  permanganate, 
turpentine,  kairin,  thallin,  following  the  inhalation  of  nitrite  of 
amyl,  ether,  etc.  It  is  encountered  also  in  hemorrhagic  transudates 
and  cystic  fluids,  and  may  occur  in  the  urine  when  methaemoglobin- 
aernia  exists. 

The  elementary  composition  of  methaemoglobin  is  the  same  as 
that  of  oxyhaemoglobin,  but  its  molecular  structure  is  manifestly 
different,  as  in  a  vacuum  it  does  not  give  up  its  oxygen.  On 
treating  with  reducing  agents  or  on  exposure  to  putrefactive  organ- 
isms, in  the  absence  of  oxygen,  it  is  converted  into  haemoglobin. 
When  oxyhaemoglobin  is  decomposed  with  dilute  acids  or  alkalies, 
methaemoglobin  is  formed  at  some  stage  of  the  process,  and  precedes 
the  formation  of  haematin.  During  the  preservation  of  oxyhaemo- 
globin in  the  dry  state,  moreover,  a  partial  transformation  into 
methaemoglobin  is  very  likely  to  occur.  The  substance  is  crystal- 
lizable,  and  may  be  obtained  in  this  form  by  treating  a  concentrated 
solution  of  oxyhaemoglobin  with  a  saturated  solution  of  potassium 
ferricyanide  until  the  color  has  changed  to  a  port-brown.  The 
mixture  is  cooled  to  0°  C,  and  treated,  with  one-quarter  of  its 
volume  of  cooled  alcohol.  When  kept  in  the  refrigerator  crystal- 
lization takes  place  in  the  course  of  a  few  days.  The  crystals  are 
of  a  brown  color,  and  occur  as  needles,  prisms,  or  hexagonal  plate- 
lets. They  may  be  purified  by  recrystallization  from  water  in  the 
presence  of  alcohol.  An  aqueous  solution  of  the  substance  is 
brown,  while  its  alkaline  solutions  are  beautifully  red.  On  exposure 
to  sunlight  its  neutral  and  dilute  solutions  gradually  assume  a 
dark-red  color,  which  is  thought  to  be  referable  to  a  transformation 
of  methaemoglobin  into  photomethcemoglobin.  On  spectroscopic 
examination  such  solutions  show  one  broad  band  of  absorption  in 
the  green  portion  of  the  spectrum  no  matter  whether  the  reaction  is 
alkaline,  neutral,  or  acid.  Methaemoglobin  proper  under  the  same 
conditions  gives  a  fairly  broad  band  of  absorption  between  C  and  D, 
nearer  C,  which  is  characteristic,  and  disappears  on  the  addition  of 
sodium  hydrate  solution.  In  addition,  two  different  bands  may  at 
times  be  seen  between  D  and  E,  which  are  thought  to  be  referable, 
however,  to  a  contamination  of  the  substance  with  haemoglobin. 
Some  observers  further  speak  of  an  additional  band  near  F,  but 
this  is  not  characteristic.  On  reduction  of  the  alkalinized  solution 
with  ammonium  sulphide  the  spectrum  of  haemochromogen  results. 


CHEMICAL   EXAMINATION  OF  THE  BLOOD.  337 

Like  oxyhemoglobin,  methsemoglobin  is  capable  of  combining 
with  certain  gases  to  form  molecular  compounds.  Of  these,  a 
carbon  dioxide  methoewioglobin,  a  methcvmoylobin  sulphide,  and  a  cyan- 
methcemoglobin  have  been  described.  Acetylene  also  is  said  to  enter 
into  combination  with  the  coloring-matter  of  the  blood.  These 
compounds,  however,  are  but  little  known.  The  methsemoglobin 
sulphide  results  when  hydrogen  sulphide  and  air  are  simultaneously 
passed  through  lake-colored  blood.  It  gives  rise  to  a  greenish-red 
color,  and  it  is  thought  that  the  greenish  discoloration  of  decom- 
posing bodies  is  referable  to  its  presence.  On  spectroscopic  exami- 
nation its  neutral  solutions  give  two  bands  of  absorption  between 
C  and  D,  of  which  one  is  brighter  and  located  near  C,  while  the 
other  and  darker  band  occupies  the  middle  portion  between  C  and 
D.  The  two  are  united  by  a  diffuse  shadow.  On  adding  a  strong 
solution  of  sodium  hydrate  the  darker  band  disappears,  and  if  now 
the  solution  is  heated  and  treated  with  a  reducing  agent,  the  spectrum 
of  hoemochromogen  results. 

The  substance  itself  has  not  been  isolated. 

Haematoporphyrin. — This  substance,  as  has  been  indicated,  results 
from  haematin  when  this  is  treated  with  concentrated  sulphuric 
acid  that  has  been  saturated  with  hydrobromic  acid.  During  this 
process  the  iron  of  the  haematin  is  split  off,  and  a  new  pigment, 
haematoporphyrin,  is  formed.  In  the  circulating  blood  of  the  verte- 
brate animals  it  is  not  found  under  normal  conditions,  but  is  appar- 
ently formed  in  certain  diseases,  and  during  the  long-continued 
administration  of  sulphonal  and  related  bodies,  as  also  in  lead 
poisoning  and  following  intestinal  hemorrhages,  when  it  may  also  be 
found  in  the  urine.  Among  invertebrate  animals  it  is  said  to  occur 
in  the  integument  of  the  star-fish,  in  certain  snails,  in  the  earth- 
worm, in  various  sponges,  etc. 

Hsematoporphyrin  is  thought  to  be  isomeric  with  bilirubin,  and  is 
thus  represented  by  the  formula  C,2Hji;N40(;.  On  reduction  it  yields 
a  pigment  which  is  possibly  identical  with  hydrobilirubin,  or  very 
closely  related  to  it.  It  may  be  obtained  in  crystalline  form  as 
a  hydrochlorate,  while  the  pigment  itself  is  amorphous.  Its  solu- 
tions in  acid  alcohol  present  a  beautiful  purple  color,  which  is 
changed  to  a  violet  blue  on  adding  an  excess  of  the  acid.  It  is 
most  conveniently  obtained  by  starting  with  haemin  and  decomposing 
tlii-i  with  glacial  acetic  acid  that  has  been  saturated  with  hydro- 
bromic acid.  Its  solutions  in  acid  alcohol  give  two  bands  of  absorp- 
tion. One  of  these  is  located  between  C  and  I),  while  the  second 
band,  which  is  much  darker  and  more  strongly  defined,  occupies 
a  position  midway  between  I)  and  E,  and  extends  as  a  shadow 
toward  I).  In  dilute  alkaline  solutions,  on  the  other  hand,  we  find 
four  band-:  one  between  C  and  D;  a  second  one,  which  is  broader 
than  the  first,  between  I)  and  E  and  about  1);  a  third  band, 
between  I>  and  E,  near  I'];  ami  finally  a  further  band  between  band 
F,  which  LS  the  widest  and  much  darker  than  the  rest.  On  treating 
22 


338  THE  BLOOD. 

with  an  alkaline  solution  of  zinc  chloride  this  spectrum  gradually 
passes  into  a  new  spectrum  with  only  two  bands,  of  which  one  is 
seen  about  D,  and  the  other  between  I)  and  E. 

Closely  related  to  haematoporphyrin,  apparently,  is  the  phyllopor- 
phyrin  which  may  be  obtained  from  the  chlorophyl  of  plants. 
From  its  formula,  C32H34jNT402,  and  that  of  haematoporphyrin  anhy- 
dride, C32H34N405,  it  is  suggested  that  both  are  different  oxidation- 
products  of  one  and  the  same  substance,  which  is  still  unknown,  but 
undoubtedly  represents  the  mother-substance  of  the  respiratory  pig- 
ment of  both  animals  and  plants.  The  spectrum  of  both  is  practi- 
cally identical.  On  distillation  with  zinc  dust  phylloporphyrin  gives 
the  pyrrol  reaction,  which  is  also  obtained  with  the  coloring-matter 
of  the  blood. 

Haematoidin. — This  pigment,  which  was  first  observed  by  Vir- 
chow  in  old  extravasations  of  blood,  in  which  it  may  occur  in  crystal- 
line form,  is  now  known  to  be  identical  with  bilirubin.  As  a  separate 
substance  it  therefore  no  longer  merits  consideration.  Its  develop- 
ment from  blood-pigment,  however,  demonstrates  the  close  relation 
existing  between  it  and  the  coloring-matter  of  the  bile  (which  see). 

I  have  pointed  out  that  in  some  of  the  lower  animals  haemoglobin 
is  also  found,  and  may  occur  in  the  blood  either  as  such  or  bound  to 
certain  cellular  elements  which  may  be  compared  to  the  red  cor- 
puscles of  the  vertebrates.  In  other  invertebrate  animals  we  find 
no  haemoglobin,  but  related  respiratory  pigments,  which  are  partly 
violet  or  purplish  red  in  color,  and  partly  blue.  The  former  com- 
prise the  so-called  floridins,  of  which  little  is  known,  while  the  latter 
group  is  represented  by  the  oxy-compound  of  haemocyanin. 

Haemocyanin  is  of  special  interest,  as  it  is  apparently  closely 
related  to  haemoglobin,  but  contains  copper  in  its  molecule  in  the 
place  of  iron.  Unlike  haemoglobin,  however,  hsemocyanin  is  itself 
colorless,  while  its  oxy-compound,  oxy haemocyanin,  presents  a  beau- 
tiful blue  color.  On  decomposition  oxyhaemocyanin  yields  an  albu- 
minous substance,  which  may  be  compared  to  globin,  and  a  copper- 
containing  pigment  which  corresponds  to  haematin.  On  reduction 
with  ammonium  sulphide  or  on  exposure  to  an  atmosphere  of  carbon 
dioxide  it  yields  the  colorless  haemocyanin.  On  spectroscopic  exami- 
nation haemocyanin  and  oxyhaemocyanin  show  a  shadow  at  both  ends 
of  the  spectrum,  which  is  more  marked  in  the  latter ;  true  absorp- 
tion-bands, however,  are  not  observed.  Neither  substance  has  been 
obtained  in  crystalline  form,  and  of  their  elementary  composition  we 
are  also  in  ignorance. 

Other  invertebrate  animals  contain  only  lipochromic  pigments  in 
their  haemolymph,  which  probably  do  not  possess  a  respiratory  func- 
tion however,  and  in  the  lowest  forms  of  life,  of  course,  special 
oxygen-carriers  are  not  required. 


CHAPTER    XV. 

THE  LYMPH. 

In  its  course  through  the  blood-capillaries  a  portion  of  the  plasma 
passes  out  through  the  vessel-walls  and  enters  a  system  of  irregular 
interfascicular  clefts,  which  are  bounded  by  bundles  of  fibrous  tissue 
and  constitute  the  radicles  of  the  lymphatic  system.  Through  these 
clefts  the  plasma  reaches  the  individual  cells  of  the  various  tissues 
and  organs  of  the  body,  and  supplies  these  with  the  requisite 
nourishment,  while  at  the  same  time  it  takes  up  the  waste  matter 
that  is  formed  in  the  metabolism  of  the  cells,  and  through  the 
lymph-vessels  carries  these  into  the  venous  current  of  the  blood. 
This  fluid-,  which  thus  contains  the  various  constituents  of  the 
blood-plasma  and  the  decomposition-products  of  the  cells,  is  termed 
the  lymph. 

In  addition  to  the  lymph-vessels  proper  and  their  radicles,  the 
lymph-clefts,  this  fluid  is  also  found  in  the  so-called  serous  cavities 
of  the  body,  viz.,  the  pleura,  the  peritoneal  and  pericardial  cavities, 
in  the  ventricles  of  the  brain  and  the  spinal  cord,  in  the  communi- 
cating subarachnoid  space,  and  also  in  the  anterior  chamber  of  the 
eye.  In  health,  however,  these  cavities  contain  but  little  fluid,  and 
quantities  sufficient  for  analytical  purposes  can  normally  be  obtained 
only  from  the  pericardial  sac,  and  at  times  from  the  subarachnoid 
space.  Under  pathological  conditions,  however,  large  accumulations 
of  fluid  may  be  observed,  and  not  only  in  the  serous  cavities  of  the 
body,  but  also  in  the  areolar  connective  tissue,  beneath  the  skin, 
and  beneath  the  muscles.  When  due  to  circulatory  disturbances,  a 
hydrsemic  condition  of  the  blood,  or  an  insufficient  elimination  of 
water  through  the  kidneys,  such  accumulations  of  fluid  are  spoken 
of  as  transudates,  while  the  term  exudates  is  applied  to  similar 
accumulations  of  inflammatory  origin. 

Formerly  it  was  supposed  that  the  lymph  resulted  from  the  blood- 
plasma  through  a  simple  process  of  lilt  ration  or  transudation  only, 
and  in  accordance  with  this  view  we  find  that  in  the  various  accumu- 
tion-  of  lymph  the  salts  and  extractives  are  presenl  in  about  the 
same  amount  as  in  the  blood-plasma.  Ileidenhain,  however,  has 
shown  that  the  flow  of  the  lymph-current  is  far  too  sluggish  to 
supply  th"  various  organs  of  the  body  with  the  proper  amount  of 
nourishment,  supposing  it-  composition  to  be  everywnere  the  same 
as  that  of  the  blood-plasma.  We  are  hence  forced  to  the  conclusion 
that  the  endothelial  cells  of  the  capillaries  possess  a  selective  secre- 

339 


340  THE  LYMPH. 

tory  power  similar  to  that  of  the  renal  epithelium,  and  are  thus 
capable  of  furnishing  to  each  tissue  its  proper  amount  and  kind  of 
food.  This  view,  however,  does  not  preclude  the  possibility  that 
some  of  the  constituents  of  the  plasma  may  pass  over  into  the  lymph 
by  a  simple  process  of  filtration,  and  it  is  likely  that  this  actually 
occurs  with  the  water  and  most  of  the  mineral  salts. 

According  to  its  origin,  then,  we  may  expect  to  find  certain  dif- 
ferences in  the  chemical  composition  of  the  lymph,  and  we  find,  as 
a  matter  of  fact,  that  such  differences  exist.  These  are,  however, 
essentially  of  a  quantitative  kind,  and  qualitatively  we  find  the  same 
constituents  in  the  lymph  from  the  various  districts  as  compared  with 
each  other  and  with  the  blood-plasma. 

Like  the  blood-plasma,  the  lymph  consists  of  a  liquid  portion,  the 
lymph-plasma,  and  cellular  elements,  the  lymph-corpuscles.  These 
latter  are  essentially  mononuclear  leucocytes,  and  are  largely  derived 
from  the  lymphoid  tissue  which  abounds  in  the  course  of  the  lymph- 
vessels.  Red  corpuscles  are  either  lacking  entirely  or  they  are  pre- 
sent in  very  small  numbers.  They  are  of  much  darker  color  than 
those  of  the  blood,  but  on  exposure  to  the  air  they  take  on  the 
bright-red  color  of  oxyhemoglobin.  According  to  some  observers, 
they  represent  transition-forms  between  the  leucocytes  and  the 
normal  red  corpuscles  of  the  blood. 

In  various  inflammatory  diseases  of  the  serous  cavities  the  leuco- 
cytes may  be  present  in  very  large  numbers,  and  in  extreme  cases, 
indeed,  they  predominate  to  such  an  extent  that  the  liquid  character 
of  the  lymph  may  be  almost  entirely  lost. 

The  appearance  of  the  lymph  is  dependent  upon  the  number  of 
the  leucocytes  and  the  amount  of  fat  present.  As  obtained  from 
fasting  animals,  it  represents  a  slightly  viscid,  straw-  or  rose-colored, 
transparent  fluid.  During  the  process  of  digestion,  on  the  other 
hand,  and  especially  after  the  ingestion  of  much  fatty  food,  it 
becomes  more  or  less  opaque,  owing  to  the  admixture  of  the  fat, 
which  is  carried  into  the  general  lymph-current  through  the  chyle, 
viz.,  the  lymph  coming  from  the  intestinal  canal. 

The  odor  of  the  lymph,  like  that  of  the  blood,  is  different  in 
different  animals.  Its  taste  is  salty,  and  the  reaction  slightly  alka- 
line.    The  specific  gravity  varies  between  1.015  and  1.021. 

The  amount  of  lymph  which  is  produced  in  the  twenty-four  hours 
is  largely  influenced  by  the  process  of  digestion.  During  starvation 
a  smaller  amount  is  thus  found  than  after  the  ingestion  of  food,  and 
it  appears,  moreover,  that  an  albuminous  diet  causes  a  much  greater 
increase  than  one  of  carbohydrates.  Active  muscular  exercise  has 
a  similar  stimulating  effect  upon  its  formation.  Artificially  the 
amount  of  lymph  can  be  increased  by  the  intravenous  injection  of 
so-called  lymphagogues,  of  which  Heidenhain  recognizes  two  classes, 
viz.,  those  which  merely  increase  the  amount  of ■ water  in  the  lymph 
and  those  which  also  bring  about  an  increase  of  the  organic  solids. 
The  former  include  such  crystalline  substances  like  sugar,  urea, 


THE  LYMPH.  341 

sodium  chloride,  etc. ;  and  Heidenhain  supposes  that  their  action  is 
dependent  upon  their  passage  into  the  lymph,  where  they  exert 
a  stimulating  effect  upon  the  cells  of  the  tissues  and  cause  an 
absorption  of  cellular  water.  The  latter,  on  the  other  hand,  are  in 
part  unknown  substances  which  can  be  extracted  from  the  muscles 
of  the  crab,  from  the  head  and  body  of  leeches,  from  the  bodies  of 
anodonts,  from  the  liver  and  intestines  of  the  dog,  and  also  com- 
prise the  peptones  and  egg-albumin.  The  influence  of  these  sub- 
stances is  apparently  exerted  upon  the  endothelial  cells  of  the 
blood-capillaries,  whereby  the  secretory  power  is  increased,  and 
we  accordingly  find  more  albumin  in  the  lymph  than  in  the  remain- 
ing blood-plasma.  Such  lymph  then,  in  contradistinction  to  the 
cellular  lymph  which  is  found  in  the  first  instance,  may  be  termed 
blood-lymph.  The  state  of  the  blood-pressure,  according  to  Heiden- 
hain, is  of  no  moment  in  bringing  about  these  changes,  and  he  found, 
as  a  matter  of  fact,  that  variations  between  10  and  20  Hgmm.  on  the 
one  hand,  and  150  to  200  Hgmm.  on  the  other  hand,  are  of  little 
influence  upon  the  amount  of  lymph  that  is  produced.  This  view, 
however,  is  in  all  probability  not  final. 

According  to  Bunge,  the  amount  of  lymph  that  is  formed  in  the 
twenty-four  hours  by  the  human  being  amounts  to  about  4000  c.c. 

A  general  idea  of  the  chemical  composition  of  lymph  may  be 
formed  from  the  following  analysis  of  Munck  and  Rosenstein. 
The  material  was  obtained  from  a  fistula  in  the  thigh  of  a  young 
woman.  Accompanying  this  is  an  analysis  of  the  blood-plasma 
(taken  from  Hammarsten)  for  comparison  : 


Lymph.  Blood-plasma. 

Water 96.5     -94.5  per  cent.  91.8  per  cent. 

Solids 3.7     -5.5  "  "  8.2    "      " 

Albumins 3.4    -4.1  "  "  6.9    "      " 

Ethereal  extract 0.06  -0.13  "  " 

Sugar       0.1  "  "  0.46  "      " 

Salts 0.8     -0.9  "  "  0.84  "      " 

Sodium  chloride    .    .    .  0.55  -0.58  "  " 

Sodium  carbonate      .    .  0.24  "  " 

Disodic  phosphate     .    .  0.028  "  " 


Of  these  constituents,  the  fat  is  subject  to  the  greatest  variations, 
and  is.  of  course,  always  more  abundant  during  the  process  of  diges- 
tion in  the  chyle  than  in  any  other  lymphatic  district.  In  Munck's 
case  the  amount  rose  to  4.7  per  cent,  after  the  ingestion  of  a  large 
amount  of  fatty  food,  and  decreased  to  0.06-0.26  per  cent,  when 
food    was   withheld    for   twenty-four  hours. 

The  (at  i-  present  in  the  lymph  to  the  greatest  extent  as  neutral 
fat,  and  it  i-  to  be  noted  that  it  bere  exists  in  a  state  of  emulsion,  so 
that  upon  microscopical  examination  the  chyle  more  especially  will 
be  seen  to  contain  innumerable  fat  droplets,  which  vary  but  little  in 
Bire  and    have  no  tendency  to  flow  together,  as  in  the  case  of  milk. 


342  TEE  LYMPH. 

Why  this  is  we  do  not  know,  but  it  has  been  suggested  that  each 
fat  droplet  is  surrounded  by  a  delicate  albuminous  envelope,  which 
is  derived  from  the  normal  albumins  of  the  lymph.  On  the  other 
hand,  it  is  conceivable  that  the  surface  layer  of  each  droplet  con- 
sists of  modified  fat  or  of  a  denser  layer  of  the  fluid  in  which  it  is 
suspended. 

The  fats  which  are  found  in  the  lymph  are  always  identical  with 
those  of  the  food,  and  can  be  recovered  for  analytical  purposes  by 
extracting  with  ether.  In  its  course  through  tissues  which  are  rich 
in  fat  no  fat  is  absorbed. 

Soaps  are  present  in  the  lymph  in  only  very  small  amounts. 

The  albumins  which  are  found  in  the  lymph  are  the  same  as  those 
of  the  blood,  and  are  present  in  the  same  ratio  to  each  other.  The 
amount  varies  somewhat  with  the  character  of  the  lymph,  but  is 
normally  always  smaller  than  that  of  the  blood-plasma.  Like  the 
blood-plasma,  so  also  does  the  lymph  coagulate  on  standing.  The 
coagula,  however,  are  very  delicate  and  tend  to  separate  out  in  frac- 
tions. Some  transudates,  indeed,  such  as  pericardial  effusions  and 
hydrocele  fluid,  do  not  coagulate  spontaneously  at  all,  while  coagula- 
tion occurs  at  once  if  leucocytes  or  blood  is  added.  The  peculiar 
behavior  of  such  lymph  is  no  doubt  due  to  the  absence  of  cellular 
elements.  It  is  to  be  noted  also  that  following  the  injection  of 
those  substances  which  prevent  coagulation  of  the  blood  coagula- 
tion of  the  lymph  similarly  does  not  occur. 

Other  albumins  besides  serum-albumin,  serum -globulin,  and  fibrin- 
ogen are  not  found  in  normal  lymph,  such  as  we  obtain  from  the 
thoracic  duct,  for  example.  Under  pathological  conditions,  how- 
ever, the  exudates  more  particularly  may  contain  the  various  albu- 
minous derivatives  of  the  leucocytes,  such  as  nucleins,  nucleo- 
albumins,  etc.  In  cysts  of  the  ovaries  and  their  appendages  met- 
albumin  or  paralbumin  may  further  be  found. 

The  amount  of  sugar  which  is  found  in  normal  lymph  is  fairly 
constant,  and  is  derived  from  the  hepatic  lymph.  It  can  be  increased 
artificially  by  ligating  the  ureters  and  then  injecting  glucose  into 
the  blood.  It  is  noteworthy  that  under  such  conditions  the  lymph 
may  contain  a  larger  percentage  of  sugar  than  the  blood  itself.  This 
further  shows  that  the  formation  of  lymph  cannot  be  explained 
upon  the  basis  of  filtration  and  osmosis  only,  and  demonstrates 
the  specific  activity  of  the  endothelial  lining  of  the  capillaries. 
Under  pathological  conditions,  it  is  claimed,  sugar  may  be  altogether 
absent. 

The  extractives  of  the  lymph  are  essentially  the  same  as  those  of 
the  plasma.  In  certain  districts,  however,  the  one  or  the  other  will 
be  found  to  preponderate,  and  in  some  localities  we  further  meet 
with  extractives  which  are  peculiar  to  that  particular  region.  In 
the  cerebrospinal  fluid,  for  example,  pyrocatechin  has  been  found  ; 
allantoin  is  present  in  the  allantoic  fluid  and  in  ascitic  accumula- 
tions ;    succinic  acid  and  inosit  may  be  obtained    from   hydrocele 


THE  LYMPH.  343 

fluid,  in  which  cholesterin  may  also  be  present  in  very  considerable 

amount.  .  .  ,  .      . 

Traces  of  urea,  uric  acid,  lecithin,  xanthm,  kreatin,  and  lactic 

acid  are  commonlv  found.  . 

Of  ferments,  we  find  a  diastatic  ferment  and  the  glucolytic  ter- 
raent  of  Lepine.  . 

The  gases  of  the  lymph  differ  from  those  of  the  blood  m  the 
presence  of  larger  amounts  of  carbon  dioxide— 35  to  45  per  cent.— 
as  compared  with  arterial  blood,  and  smaller  amounts  than  are 
found  in  venous  blood.  The  tension  of  the  carbon  dioxide,  as 
compared  with  venous  blood,  is  lower,  which  suggests  that  a  por- 
tion of  the  gas  enters  the  blood  from  the  lymph  through  a  reverse 
secretorv  activity  of  the  capillary  endothelium.  Oxygen  is  present 
only  in 'traces,  while  the  amount  of  nitrogen  is  the  same  in  both,  viz., 
1.6  per  cent. 

For  purposes  of  comparison  and  reference,  a  few  analyses  ot  some 
of  the  more  important  normal  and  pathological  varieties  of  lymph 
are  appended.  Some  of  these  will  be  considered  in  greater  detail 
in  subsequent  chapters. 

Analysis  of  Human  Pericardial  Fluid  (Hammarsten). 
Water *%*» 


Solid 


39.14 


Albumins 2^° 

Fibrin V^i 

(ilobulins      • kg 

Serum-albumin "no 

Extractives      --^ 

Soluble  salts     .    . _ °™ 

Sodium  ebloride '■-- 

Insoluble  salts °-10 


Analysis  of  Dog's  Chyle  (Hoppe-Seyler). 

Water 906.77 

Solids ^.23 

Fibrin }•" 

Albumins  and  jrlobulms -i.uo 

lilthin  .     I 64.8G 

<  ii'.lcsterin  j 

Fatty  acids  and  soaps  1                                  2.34 

Other  organic  bodies  j 

Mineral  -alts '••'"- 


Anai.v-i-   OF    AqTTBOUS   HlTMOB  OF  CAM?   (Halliburton). 
Water  986.87 

&£  • : : : i«> 

Albumins ,    ,7 

Extractives       4.21 

Inorganic  salts '■'" 

-  dium  chloride     °-8y 


344  THE  LYMPH. 

Analysis  of  Cerebrospinal  Fluid  (Gautier). 

Water 987.00 

Solids 10.59 

Albumins      1.10 

Fats 0.09 

Cholesterin 0.21 

Alcoholic  and  aqueous  extracts,  minus  salts,  but  including 

sodium  lactate 2.75 

Salts      6.44 

Chlorides 6.14 

Sulphates      0.20 

Earthy  phosphates      0.10 

Ammonia traces. 

Analyses  of  Pleural  Effusions  (Gautier). 

Acute 
pleurisy. 

Water 937.60 

Organic  matter 54.40 

Fibrin      0.09 

Mineral  matter 8.00 


Chronic             C 

Hydrothora 

pleurisy. 

(cardiac). 

933.80 

958.70 

58.20 

32.30 

0.00 

0.19 

8.00 

9.00 

•rivon  and  Sch> 

erer) . 

Chronic 

Hepatic 

nephritis. 

cirrhosis. 

978.00 

9S4.50 

Ovarian 
cancer. 

Water 946.50 

Serum-albumin 19.40 

Serum-globulin      18.581  Q  4A                    ftl7 

Mucin  (?) 0.95/  8>4U                    b-1' 

Fats              1  ,  q    . 

Cholesterin   [ 1.25  *' 00  }                  '      ' 


Extractives 

Soluble  salts 5.521 

Insoluble  salts 7.53  J 


1.9 
.0C 

.00  8.46 


Analysis  of  Hydrocele  Fluid  (Hammarsten). 

Water 938.85 

Solids 61.15 

Fibrin 0.59 

Globulins      13.25 

Serum-albumin 35.94 

Ethereal  extract 4.02 

Soluble  salts 8.60 

Insoluble  salts      0.66 

Analysis  of  Amniotic  Fluid  (Labroche). 

Human. 

Water 987.300 

Solids 16.700 

Serum-albumin 2.590 

Mucin         1 

Albumoses  j- 1.604 

Glucose      J 

Urea      0.450 

Fats 0.356 

Mineral  salts 7.695 

Sodium  chloride      6.071 

Disodium  phosphate 1.621 

Sulphates traces. 

Salts  of  calcium  and  potassium none. 


Til E  LYMPH.  345 

j 

Analysis  of  Lymph  from  Cellular  Tissue  ((Edema). 

Water 975.20 

Albumins      5.42 

Fats  and  extractives 3.76 

Mineral  salts * 15.62 

Analysis  of  Pus. — The    composition  of  the   leucocytes   which  v 
enter  into  the   formation   of  pus   has   been  considered   (pages  303 
and  322).     An  analysis  of  pus-serum  is  here  given,  which  is  taken 
from  Robin  : 

Water 937.90 

Metalbumin        ~) 

Serum-albumin  [■ 11.00 

Serum-globulin  J 

Lecithin 6.00 

Fats  and  soaps 10.00 

Cholesterin       3.50 

Serolin      1.00 

Leucin,  tyrosin,  and  extractives  in  general 15.00 

Salts  of  organic  acids traces. 

Sodium  chloride      3.11 

Sodium  phosphate traces. 

Phosphates  of  calcium  and  magnesium      0.50 

Sulphates 1.87 

Salts  of  iron  and  silica      0.16 

THE  SYNOVIAL  FLUID. 

The  synovial  fluid,  though  not  a  lymph  in  the  narrower  sense  of 
the  term,  is  for  convenience'  sake  briefly  described  at  this  place.  It 
is  the  specific  secretion  of  the  synovial  membrane  of  the  joints, 
and  constitutes  a  strongly  alkaline,  viscid,  yellowish,  somewhat 
cloudy  fluid.  In  addition  to  the  common  albumins,  fats,  salts,  and 
extractives  of  the  lymph,  it  contains  also  a  peculiar  mucinous  body, 
which  is  termed  synovin,  and  apparently  belongs  neither  to  the 
nucleo-albumins  nor  the  mucins  or  mucoids.  It  can  be  precipitated 
with  acetic  acid  and  coagulates  on  the  application  of  heat.  Of  its 
nature,  however,  nothing  further  is  known.  In  addition,  another 
mncin-like  body  is  found  which  is  rich  in  phosphorus,  and  probably 
belongs  to  the  ouoleo-albumins. 

Quantitatively  the  composition  of  the  synovial  fluid  varies  with 
exercise  and  pest  in  such  a  manner  that  on  motion  the  mucinous 
body,  as  also  the  albumins  and  extractives,  increase,  while  the  salts 
diminish.  This  is  shown  in  the  appended  analyses,  which  are  taken 
from   Frerichs  : 


Stalled  ox. 


Ox  on 
pasture. 

Water 960.90  948.54 

Solids 30.10  51.50 

Mucin  (?)     2.40  5.60 

Albumins  and  extractives 15.76  35.12 

0.62  0.76 

11.32  9.98 


CHAPTER     XVI. 

THE  MUSCLE-TISSUE. 

I  have  pointed  out  that  while  in  the  monocellular  organisms  the 
various  functions  of  the  body  are  carried  on  by  the  single  cell,  a 
gradual  division  of  labor  occurs  as  we  ascend  in  the  scale  of  both 
animal  and  vegetable  life,  where  groups  of  cells  are  set  aside  for  the 
performance  of  certain  special  functions.  Structurally  this  division 
of  labor  finds  its  expression  in  a  more  or  less  well-marked  deviation 
from  the  original  type,  as  has  been  shown.  At  first  sight,  it  is. 
thus  difficult  to  connect  the  highly  differentiated  muscle-cell  with 
the  apparently  much  more  simple  ovum  from  which  it  has  originated. 
The  element  of  reproduction  and  secretion  is  here  manifestly  placed 
in  the  background,  while  in  its  co-ordinate  and  rapid  contraction  on 
stimulation  we  have  abundant  evidence  of  its  highly  specialized 
function.  That  this  should  further  be  expressed  in  the  chemical 
composition  of  the  cells  suggests  itself  at  once.  As  a  matter  of 
fact,  we  here  find  substances  which  may  be  regarded  as  specific 
muscle  components,  and  it  seems  warrantable  to  assume  that  a 
definite  connection  exists  between  these  bodies  and  the  special  func- 
tion of  the  cell.  On  chemical  examination  we  may  then  further 
expect  to  meet  with  the  various  products  of  katabolism,  so  far  as 
these  are  found  in  the  muscle-tissue  proper,  and  have  not  as  yet  been 
removed  by  the  blood  or  the  lymph. 

Before  proceeding  to  a  study  of  these  various  substances  in  detail, 
a  few  analyses  of  muscle-tissue  are  here  introduced,  which  will 
furnish  a  general  idea  of  its  chemical  composition.  Qualitatively 
this  is  fairly  constant,  but  quantitative  variations  occur  which  are 
often  very  marked. 

In  preparing  the  tissue  for  analytical  purposes,  the  blood  should 
first  be  washed  out  entirely  with  dilute  saline  solution  (0.6  per  cent.). 
Fibrous  tissue  and  fat  must  be  dissected  away  as  far  as  possible  and 
all  larger  bloodvessels  removed.  The  material  is  then  further 
scraped,  so  as  to  get  rid  of  as  much  of  the  connective  tissue  as  pos- 
sible which  binds  the  individual  fibres  together,  and  is  now  ready 
for  examination. 

Analyses  of  Fresh  Muscle-tissue  (Neumeister). — The  figures 
represent  average  values,  which  have  been  collected  from  various 
sources,  and  have  reference  to  mammalian  muscle-tissue  in  general, 
unless  otherwise  stated. 

346 


THE  MUSCLE-ALBUMINS.  347 

Per  cent. 

Water *. 75.50 

Solids 24.50 

Organic  constituents 23.50 

Myosin  (?) 7.74 

Xncleins 0.37 

Albumins,  proteids,  and  albuminoids  (insoluble  in 

neutral  solution) 15.25 

Collagen    (referable  to  interiibrillary   connective 

tissue) 3.16 

Fats 3.71 

Glycogen 0.7-1.0 

Lactic  acid 0.1-1.0 

Inosit 0.003 

Kreatin 0.21-0.28 

Xanthin 0.01-0.11 

Hypoxantbin 0.04-0.12 

Guanin      0.005 

Inorganic  constituents 1.000 

Phosphoric  acid 0.4674 

Chlorine 0.0672 

Potassium  oxide 0.4654 

Sodium  oxide 0.0770 

Calcium  oxide 0.0086 

Magnesium  oxide 0.0412 

Iron  oxide 0.0057 

In  studying  this  analysis  we  observe  that,  aside  from  the  mineral 
constituents,  various  bodies  are  encountered  here  which  represent 
distinct  products  of  katabolism,  and  which  are  hence  most  likely  not 
concerned  in  the  specific  function  of  the  muscle-tissue.  These  com- 
prise the  common  extractives,  viz.,  xanthin,  hypoxanthin,  guanin, 
kreatin,  lactic  acid,  and  possibly  also  inosit.  They  are  no  doubt 
formed  as  a  consequence  of  cellular  activity,  but  play  no  role  in  the 
function  of  the  muscle  proper.  On  the  other  hand,  we  meet  with 
food-stuffs  proper,  viz.,  albumins,  carbohydrates,  and  fats,  and  we 
may  a  priori  expect  that  all  these  substances,  conjointly  or  in- 
dividually, are  directly  concerned  in  the  contractile  function  of 
the  cell. 

THE   MUSCLE-ALBUMINS. 

An  in  the  case  of  all  tissues  of  the  body,  the  muscle-tissue  also 
consists  of  a  liquid  portion,  the  so-called  muscle-plasma,  and  a  more 
solid  portion,  which  may  be  termed  the  muscle-stroma. 

Muscle-plasma  may  be  obtained  in  the  following  manner :  while 
the  animal  is  -till  living  the  blood  is  thoroughly  washed  from  the 
large  skeletal  muscles  by  injecting  into  the  larger  arteries  a  dilute 
saline  solution  that  has  been  wanned  to  the  temperature  of  the  body, 
and  allowing  the  fluid  to  escape  from  the  corresponding  veins.    This 

i-  continued  after  death  until  the  outflowing  water  is  colorless.     The 

muscles  are  then  rapidly  dissected  oil',  ground  to  a  pulp  together 
with  pumice-stone, and  passed  through  a  filter-press.  The  resulting 
liquid  i-  tli'-  muscle-plasma. 

1  In  birds 


348  THE  MUSCLE-TISSUE. 

While  this  procedure  is  applicable  in  the  case  of  mammalian 
muscle-tissue  in  general,  special  precautions  are  necessary  if  the 
muscles  of  the  lower  animals  are  to  be  studied.  In  the  frog,  for 
example,  the  tissue,  after  removal  of  the  blood,  must  be  frozen  in 
a  gradual  manner,  after  which  the  entire  process  is  continued  at 
a  temperature  below  — 3°  C.  A  snow-like  mass  is  finally  obtained, 
which  melts  at  — 3°  C. 

The  color  of  the  muscle-plasma  varies  with  the  color  of  the 
muscles  from  which  it  has  been  obtained.  In  the  case  of  the  dog  it 
is  of  a  brownish  color,  in  rabbits  it  is  yellowish  red,  and  in  frogs 
a  light  yellow.  The  particular  shade  of  color,  as  will  be  seen  later, 
is  a  direct  expression  of  the  degree  of  functional  activity  of  the 
individual  muscles,  and  is  in  part  due  to  haemoglobin  and  in  part  to 
certain  lipochromes. 

The  reaction  of  the  plasma  is  neutral  or  slightly  alkaline. 

On  standing  for  a  length  of  time  mammalian  muscle-plasma 
gradually  undergoes  a  process  of  coagulation,  but  it  is  to  be  noted 
that  the  coagulum  M-hich  separates  out  is  slight  in  amount.  In 
the  case  of  the  frog,  on  the  other  hand,  the  entire  bulk  of  the 
plasma  becomes  gelatinous,  and,  in  contradistinction  to  the  mam- 
malian plasma,  this  process  begins  at  a  temperature  of  0°  C.  As  in 
the  case  of  the  blood-plasma,  the  coagulum  gradually  contracts,  and 
the  liquid  which  remains  is  then  spoken  of  as  muscle-serum.  The  reac- 
tion is  then  acid.  The  substance  which  composes  the  clot  is  termed 
my og en-fibrin.  The  behavior  of  muscle-plasma  is  thus  quite  simi- 
lar to  that  of  blood-plasma,  and  here,  as  there,  the  resulting  fibrin 
is  derived  from  an  albuminous  substance  which  was  previously  pre- 
sent in  solution.  This  substance  is  here  termed  myogen.  In  addi- 
tion the  muscle-plasma  contains  another  albuminous  body,  myosin  ; 
and  it  appears  from  the  researches  of  v.  Furth  that  these  two  sub- 
stances are  the  only  soluble  albumins  which  are  contained  in  muscle- 
tissue,  if  we  disregard  a  variable  amount  of  a  soluble  myogen-fibrin, 
which  is  itself  a  derivative  of  myogen. 

Myogen. — Isolation. — Myogen  is  most  conveniently  obtained 
from  muscle-plasma  after  the  myosin  has  been  removed  by  the 
previous  addition  of  ammonium  sulphate  to  the  extent  of  28  per 
cent.  The  resulting  precipitate  is  removed  and  the  filtrate  saturated 
with  the  same  salt  in  substance.  The  myogen  is  thus  thrown  down 
together  with  the  soluble  myogen-fibrin.  It  is  washed  with  a  satu- 
rated solution  of  ammonium  sulphate,  dissolved  in  water,  and  freed 
from  the  soluble  myogen-fibrin  by  heating  to  40°  C,  when  this  is 
transformed  into  the  insoluble  form  and  is  filtered  off.  The  remain- 
ing solution  contains  the  myogen  in  pure  form. 

In  its  general  properties  it  resembles  the  albumins  proper,  in  con- 
tradistinction to  the  globulins.  It  is  soluble  in  water,  and  can  be 
precipitated  by  alcohol,  by  salting  with  ammonium  sulphate,  sodium 
chloride,  and  magnesium  sulphate.  The  two  latter  salts,  however, 
do   not   cause   a   complete   precipitation.     Alcohol   (92   per   cent.) 


THE  M I  'SCLE-A  LB  CMIXS.  34  9 

renders  tbe  substance  insoluble  to  a  slight  extent,  but  the  greater 
portion  is  refractory  in  this  respect. 

Bv  acetic  acid  myogen  is  precipitated  only  in  the  presence  of  a 
neutral  salt,  but  redissolves  in  an  excess  of  the  acid,  with  the  forma- 
tion of  syntonin.  The  tendency  to  the  transformation  into  albu- 
minates is  indeed  more  marked  in  the  case  of  the  soluble  muscle- 
albumins,  in  general,  than  with  any  other  forms.  Mineral  acid-  are 
in  this  respect  still  more  active  than  acetic  acid,  and  as  a  conse- 
quence a  precipitation  of  myogen  is  observed  only  when  such  acids 
are  present  in  certain  proportion. 

Carbonic  acid  and  the  salts  of  the  heavy  metals  precipitate  myo- 
gen only  in  the  presence  of  a  neutral  salt. 

Myogen  coagulates  at  a  temperature  of  from  55°  to  65°  C.  It  is 
not  precipitated  on  dialysis. 

When  solutions  of  myogen  are  kept  at  a  certain  temperature,  and 
in  the  presence  of  a  definite  amount  of  a  neutral  salt,  the  substance 
is  gradually  transformed  into  a  soluble  form  of  myogen-fibrin,  which 
differs  from  myogen  in  the  fact  that  it  is  throwwdown  on  dialysis, 
and  in  its  point  of  coagulation,  which  lies  at  40°  C.  As  has  been 
stated,  a  certain  amount  of  soluble  myogen-fibrin  seems  to  occur  pre- 
formed in  the  muscle-tissue,  and  separates  out  gradually  on  standing. 
At  40°  C,  however,  this  occurs  instantaneously.  By  coagulation 
the  soluble  myogen-fibrin  is  transformed  into  the  insoluble  form,  the 
myogen-fibrin   pr<  >per. 

The  relative  amount  of  myogen,  as  compared  with  myosin  (see 
below),  which  is  found  in  muscle-tissue  varies  in  all  probability  with 
different  animals.  In  rabbits,  v.  Fiirth  observed  that  myosin  repre- 
sented about  80  per  cent,  of  the  total  amount  of  soluble  albumin-. 

The  amount  of  soluble  myogen-fibrin  which  is  included  in  the 
above  figures  is  in  mammals  apparently  very  small,  as  only  slight 
coagula  are  formed  when  the  plasma  is  heated  to  40°  C.  But  in  the 
frog  large  amounts  are  manifestly  present.  In  some  animals,  on  the 
other  hand,  it  is  apparently  absent. 

Myosin. — Myosin  i-  conveniently  isolated  from  muscle-plasma  by 
salting  with  ammonium  sulphate  to  the  extent  of  2X  per  cent. 
Sodium  chloride  and  magnesium  sulphate  may  also  be  employed, 
but  it  i-  then  necessary   to  add  the  salt  to  saturation. 

Tli"  substance  is  a  globulin,  and,  curiously,  contains  a  consider- 
able amount  of  calcium.  It  is  soluble  in  dilute  saline  solution-,  and 
i-  precipitated  from  these  solutions  by  Baiting,  as  just  indicated,  by 
passing  n  stream  of  carbon  dioxide  through  its  solutions,  by  diluting 
with  water,  and  on  dialysis.  It  i-  characterized  bv  its  pronounced 
tendency  to  coagulate,  ami,  unlike  myogen,  i-  rendered  almost 
entirely  insoluble  on  precipitation  with  alcohol.  Like  tlii-.  it  i<  also 
readily  transformed  into  syntonin  or  alkaline  albuminate  on  treating 
with  acids  or  alkalies  ;  and  here,  as  there,  a  precipitation  results 
only  if  very  dilute  acids  are  used.  In  an  excess  the  precipitate 
rapidly  dissolves.     On  heating  solutions  of  myosin  to  35     ( '.  1 1 1<- 


350  THE  MUSCLE-TISSUE. 

substance  is  gradually  coagulated,  while  this  occurs  at  once  at  a  tem- 
perature of  50°  C. 

In  its  insoluble  form  myosin  is  termed  myosin-fibrin,  which,  like 
the  insoluble  myogen-fibrin,  belongs  to  the  class  of  the  coagulated 
albumins. 

Of  special  interest,  further,  is  the  fact  that  on  evaporating  a  few 
drops  of  a  solution  of  myosin  in  soda  solution  on  a  slide,  at  a  low 
temperature,  a  jelly-like  material  is  obtained,  which  on  polariscopic 
examination  is  seen  to  be  doubly  refracting.  In  this  respect  it 
behaves  exactly  as  the  anisotropic  material  which  is  found  in  the 
dark  bands  of  the  voluntary  muscle-fibres. 

The  amount  of  myosin  found  in  the  muscle-tissue  of  the  rabbit  is 
much  less  than  that  of  myogen,  and,  according  to  v.  Furth,  corre- 
sponds to  only  20  per  cent,  of  the  total  amount  of  soluble  albumins. 

Significance  of  the  Common  Muscle-albumins. — Of  the  part 
which  the  common  muscle-albumins  take  in  the  function  of  the  cell 
little  is  known  that  is  definite.  From  the  researches  of  some  ob- 
servers, it  appears  that  the  nitrogenous  components  of  the  albumins, 
at  least,  do  not  furnish  the  energy  which  is  here  required.  Petten- 
kofer  and  Voit  have  thus  shown  that  an  increase  in  the  amount  of 
muscular  work  does  not  lead  to  an  increased  elimination  of  nitrogen 
or  to  an  increase  which  is  insignificant.  This  view  is  now  gener- 
ally held ;  but  it  must  be  admitted  that  evidence  is  not  lacking 
which  suggests  that  an  increased  albuminous  destruction  may  occur 
nevertheless  when  the  amount  of  work  is  increased.  It  has  been 
shown,  as  a  matter  of  fact,  that  the  total  elimination  of  sulphur, 
which  usually  follows  that  of  the  nitrogen  quite  closely,  is  increased 
by  muscular  exercise  and  diminished  thereafter.  Bat  while  we  may 
admit  that  the  nitrogenous  components  of  albumin  may  furnish  a 
certain  fraction  of  the  energy  which  is  required  in  muscular  work, 
this  is,  after  all,  but  slight,  and  there  is  abundant  evidence  to  show 
that  by  far  the  greater  amount  of  energy  must  be  referable  to  the 
decomposition  of  non-nitrogenous  material. 

The  question,  of  course,  suggests  itself,  Do  the  soluble  albumins 
of  the  muscle-plasma  represent  the  contractile  element  of  the 
muscle-tissue?  but  to  this  question  no  answer  can  as  yet  be  given. 
We  might  imagine  that  in  some  manner  a  transformation  of  the 
soluble  albumins  into  the  fibrin  form  occurs,  and  vice  versa  ;  but  of  this 
we  have  no  evidence  in  the  living  tissue.  On  the  other  hand,  we 
know  that  rigor  mortis,  as  well  as  the  rigor  which  results  from  ex- 
posure of  muscle-tissue  to  a  temperature  of  47°  C,  is  owing  to  such  a 
change,  and  it  is  quite  probable  that  in  either  event  both  myosin  and 
myogen  pass  over  into  the  coagulated  state.  The  subsequent  relaxa- 
tion is  then  no  doubt  referable  to  the  formation  of  syntonin,  which 
may  be  effected  by  the  lactic  acid  that  is  then  ahvays  found  in  consid- 
erable amount,  and  may  be  aided  by  the  presence  of  certain  ferments. 

Whether  or  not  a  myosin-ferment  exists  which  is  responsible  for 
the  transformation  of  the  soluble  albumins  into  the  insoluble  form, 


THE  MUSCLE-ALBUMINS.  351 

has  not  been  ascertained,  but  is  very  probable.  Some  writers  indeed 
regard  the  coagulation  of  muscle-plasma  which  occurs  on  standing  as 
being  referable  to  the  presence  of  such  an  enzyme. 

Other  Albumins. — Besides  myosin  and  myogen,  which  latter  was 
formerly  termed  myosinogen,  muscle-plasma  was  also  supposed  to 
contain' traces  of  serum-albumin,  myoglobulin,  and  myo-albumose. 
v.  Fiirth,  however,  has  shown  that  any  trace  of  serum-albumin  that 
may  be  found  is  referable  to  the  presence  of  small  amounts  of  lymph 
or  blood  that  have  not  been  removed  by  washing,  and  that  if  this  is 
dune  with  special  care  no  serum-albumin  can  be  demonstrated. 
Halliburton's  myoglobulin,  moreover,  he  regards  as  identical  with 
myogen,  while  the  existence  of  a  myo-albumose  in  muscle-plasma 
has  been  disproved  by  more  recent  investigations.  That  substances 
belonging  to  the  albumoses  may  be  found  in  muscle-tissue  after  death, 
when  syntonin  also  is  found,  is,  of  course,  likely,  but  in  the  living 
tissue  their  presence  can  hardly  be  expected  under  normal  conditions. 

Myoproteid  is  a  substance  which  v.  Fiirth  obtained  from  the 
muscle-plasma  of  fish.  Of  its  chemical  nature,  however,  nothing 
further  is  known  than  the  fact  that  it  apparently  does  not  belong  to 
the  commonly  recognized  classes  of  albumins.  It  is  neither  a 
nucleo-albumin  nor  a  glucoproteid. 

Nucleins  are  not  found  in  the  muscle-plasma,  but  can  be  isolated 
from  the  muscle-tissue  as  a  whole  or  from  the  insoluble  material 
which  remains  in  the  filter-press  after  separation  from  the  plasma. 
Their  amount  is  small,  and  in  accordance  with  the  slight  degree  to 
which  the  nuclei  enter  into  the  structural  composition  of  the  muscle- 
cell.  Larger  amounts  arc  obtained  from  embryonic  muscle,  where 
cellular  reproduction  is,  of  course,  more  active.  From  the  tissue  of 
an  adult  dog  Pekelharing  obtained  about  0.37  per  cent.  These 
nucleins  must  be  regarded  as  the  material  from  which  the  xanthin- 
bases  that  can  always  be  demonstrated  in  muscle-tissue  are  derived. 
These  will  be  considered  in  detail  later. 

Phosphor-carnic  Acid. —  A  few  years  ago  Siegfried  announced 
that  after  removing  the  phosphates  from  extracts  of  muscle-tissue, 
and  treating  with  ferric  chloride,  under  the  application  of  heat,  a 
phosphorus-containing  iron  compound  is  obtained,  which  is  insolu- 
ble in  water,  but  easily  soluble  in  solutions  of  the  alkalies.  This 
~nl )~t an**-  he  regards  as  the  irou  salt  of  an  organic  acid,  which  he 
term-  phosphor-carnic  acid  ;  the  salt  he  speaks  of  as  carniferrin. 
On  decomposition  with  barium  hydrate  he  then  obtained  tin;  barium 
-alt  of  ;i  cry-tiilli/able  acid,  ctiriiic  <n-id,  to  which  he  gives  the 
formula  (', J\r  SO.      In    addition,  phosphoric    acid,    carbonic   acid, 

paralactic  acid,  succinic  acid,  and  a  substance  which  apparently 
belongs  to  the  carbohydrate  group,  are  found.  In  his  more  recent 
communications  Siegfried  expresses  the  opinion  that  his  carnic  acid 
i-  in  reality  pure  antipeptone.  This  question  is  still  under  debate, 
and  i-  strongly  combated  by  Kutscher  and  others.  Kutscher, 
indeed,  bae  shown  that   Kiihne's  antipeptone  is  in  reality  a  mixture 


352  THE  MUSCLE-TISSUE. 

of  'different  substances,  and  he  has  demonstrated  that  it  can  be  sepa- 
rated into  two  fractions,  one  of  which  is  precipitated  by  phospho- 
tungstic  acid,  while  the  other  remains  in  solution.  In  the  first 
fraction  he  then  demonstrated  the  presence  of  hexon-bases,  while  in 
the  second  portion  mono-amido-acids  were  found.  Thus  far,  how- 
ever, only  a  fraction  of  the  antipeptone  has  been  resolved  into  sev- 
eral components,  and  we  must  admit  that  there  is  no  evidence 
to  show  that  the  remaining  portion  may  not  be  represented  by  a 
single  substance.  Whether  or  not  this  unresolved  portion  is  identical 
with  Siegfried's  carnic  acid,  however,  remains  to  be  seen.  If  so,  the 
remarkable  fact  would  be  demonstrated  that  a  peptone  may  occur  in 
crystalline  form. 

As  regards  the  significance  of  his  phosphor-carnic  acid,  Siegfried 
expresses  the  opinion  that  it  may  serve  as  one  of  the  sources  of 
muscular  energy,  and  he  points  out  that  in  the  working  muscle  car- 
bonic acid  must  of  necessity  be  formed  on  hydrolysis  of  phosphor- 
carnic  acid  even  though  oxygen  be  absent.  In  this  manner  the 
observation  of  Hermann  would  be  explained,  viz.,  that  a  bloodless 
muscle  can  still  work  for  a  while  in  the  absence  of  oxygen  and  give 
off  carbon  dioxide.  The  lactic  acid  and  phosphoric  acid  which 
are  also  known  to  be  set  free  during  muscular  activity,  Siegfried 
likewise  refers,  in  part  at  least,  to  a  hydrolytic  decomposition  of  his 
phosphor-carnic  acid.  The  question,  however,  whether  the  carbo- 
hydrate and  phosphoric  acid  group  only  are  liberated,  he  leaves 
undecided. 

Of  the  chemical  nature  of  phosphor-carnic  acid  little  is  known ; 
but  it  is  manifestly  closely  related  to  the  nucleins,  and  is  accordingly 
termed  a  nucleoli. 

Ferments. — The  ferments  which  occur  in  muscle-tissues  have 
received  but  little  consideration.  Several  varieties  apparently  exist. 
It  has  thus  been  shown  that  a  pepsin,  ptyalin,  and  maltase  are  present, 
and  it  seems  probable  that  a  myosin  ferment  and  a  lactic-acid-form- 
ing enzyme  further  exist.  The  two  latter,  however,  have  not  been 
isolated.  The  former  are  generally  regarded  as  being  derived  from 
the  digestive  glands,  and  it  is  supposed  that  they  have  found  their 
way  into  the  muscle-tissue  more  or  less  accidentally.  I  have  pointed 
out,  however,  that  no  cogent  reason  exists  for  regarding  the  various 
ferments  which  are  found  in  the  organs,  and  which  in  their  general 
behavior  resemble  the  digestive  ferments,  as  identical  with  these, 
and  I  have  no  doubt  that  future  researches  will  show  that  they  play 
an  important  role  in  the  metabolism  of  the  tissues  of  the  body.  To 
assume  that  the  chymosin  which  is  found  in  the  urine  must  be  iden- 
tical with  the  chymosin  of  the  gastric  juice,  on  the  basis  that  its 
formation  in  the  kidneys,  for  example,  would  lack  an  adequate 
explanation,  seems  to  me  unwarrantable.  For  we  know  that  a 
milk-curdling  ferment  is  not  peculiar  to  the  mammalian  organism, 
but  may  occur  even  in  plants. 

Muscle-Stroma. — Of  the  chemical  nature  of  the  so-called  muscle- 


GLYCOGEN.  353 

stroma,  which  remains  after  the  extraction  of  the  soluble  albumins 
with  a  5  per  cent,  solution  of  ammonium  chloride,  we  kuow  only 
that  the  material  in  question  consists  of  an  albuminous  substance. 
It  is  apparently  a  native  albumin,  and,  like  the  soluble  muscle- 
albumins,  characterized  by  the  ease  with  which  it  is  transformed  into 
alkaline  albuminate  on  treating  with  dilute  solutions  of  alkalies. 

According  to  Danilewski  and  Holmgren,  the  structure  of  the 
muscle-fibre  is  in  no  ways  altered  by  dissolving  out  the  soluble 
albumins,  and  it  would  thus  appear  that  the  stroma  represents  the 
actual  contractile  substance  of  the  tissue.  Whether  or  not  this  is 
actually  the  ease,  however,  is  as  yet  unknown. 

The  sarcolemma  apparently  consists  of  a  substance  which  belongs 
to  the  albuminoids,  and  resembles  elastin  in  its  general  properties. 

THE   MUSCLE-PIGMENTS. 

A-  I  have  already  indicated,  the  color  of  the  muscle-plasma  is  dif- 
ferent in  different  animals,  and  practically  coincides  with  the  color 
of  the  muscle-tissue  itself.  In  some  animals,  and  notably  the  mam- 
mals, this  is  dark  red,  while  the  muscles  of  others  are  almost  color- 
less. But  even  in  those  vertebrate  animals  in  which  no  color  is 
observed  in  the  skeletal  muscles  as  a  whole  the  heart-muscle  and 
the  diaphragm  always  appear  dark  red.  This  difference  is  thought 
to  depend  upon  the  degree  of  activity  of  the  different  muscles,  but 
apparently  has  nothing  to  do  with  the  velocity  of  contraction  of 
which  a  muscle  is  capable. 

The  red-muscle  pigment  proper  is  now  known  to  be  identical 
with  the  haemoglobin  of  the  blood,  and  probably  serves  the  same 
purpose,  as  a  carrier  of  oxygen,  in  the  internal  respiration  of  the 
tissue.  That  it  actually  occurs  within  the  cells  is  now  undoubted. 
Curiously  enough,  the  same  pigment  is  found  in  the  red  muscles  of 
certain   insects,  in   which   no  haemoglobin  otherwise  occurs. 

Jn  addition  to  haemoglobin  various  lipochromes  may  also  be 
encountered  in  muscle-tissue,  and  are  especially  abundant  in  certain 
fishes,  such  as  the  salmon  and  the  sea  trout.  Of  their  origin  and 
significance  nothing  is  known. 

GLYCOGEN. 

The  glycogen  which  is  found  in  muscle-tissue  does  not  occur  in 
the  body  of  the  cells  proper,  butia  distributed  between  the  individual 
fibre-  in  the  form  of  fine  threads,  which  are  apparently  connected  with 
the  connective-tissue  corpuscles, 

The  substance  is  formed  synthetically  in  the  muscle-tissue  through 
a  polymerization  of  the  anhydride  radicles  of  glucose,  which  is 
carried  to  the  tissue  either  directly  from  the  intestinal  tract,  or  which 
results  from  the  hepatic  glycogen  through  a  process  of  depoly- 
merization.     Thai  the  muscle-tissue  is  in  fict  capable  of  effecting  this 

2.". 


354  THE  MUSCLE- TISSUE. 

synthesis  is  now  undoubted.  It  has  thus  been  shown  that  in  frogs 
a  deposition  of  glycogen  occurs  following  the  subcutaneous  injection 
of  a  solution  of  glucose,  even  after  removal  of  the  liver.  The 
amount  of  glycogen  which  is  deposited  in  the  muscle-tissue  probably 
represents  about  one-half  of  the  total  amount  that  is  found  in  the 
entire  body,  and  in  man  corresponds  to  150  grammes.  It  repre- 
sents the  most  important  source  of  energy  which  is  at  the  disposal 
of  the  tissue,  and  is  constantly  consumed,  even  when  the  muscle  is 
at  rest.  This  is  apparent  from  the  fact  that  after  section  of  the 
nerves  more  glycogen  is  found  in  a  given  muscle  than  in  the  cor- 
responding muscle  of  the  other  side,  while  ordinarily  this  is  the 
same.  While  at  work  the  consumption  of  glycogen  increases,  and 
after  a  comparatively  short  time  already  the  substance  has  entirely 
disappeared.  If  now  a  period  of  rest  follows,  glycogen  is  again 
stored  in  the  muscle,  and  so  on.  It  is  to  be  noted,  however,  that 
the  working  muscle  is  constantly  taking  up  sugar  from  the  blood, 
which  in  turn  is  derived  from  the  glycogen  of  the  liver,  and  that 
its  function  may  continue  even  though  a  deposition  of  glycogen,  as 
such,  does  not  occur.  This  shows  that  the  muscle  glycogen,  like 
that  of  the  liver,  is  in  reality  a  reserve  food,  and  is  here  deposited 
for  immediate  use.  In  starving  animals  it  gradually  disappears, 
but,  in  contradistinction  to  the  glycogen  of  the  liver,  its  supply  is 
not  exhausted  until  the  liver  itself  is  free  from  glycogen. 

The  chemical  changes  which  are  involved  in  the  transformation  of 
glycogen  into  glucose  are  probably  the  same  as  those  which  occur 
during  the  process  of  digestion.  Erythrodextrin  thus  first  results, 
and  is  then  transformed  into  achroodextrin,  and  this  into  maltose, 
which  in  turn  is  inverted  to  glucose.  This  is  finally  decomposed, 
with  the  formation  of  carbon  dioxide  and  water.  The  amount  of 
carbon  dioxide  that  is  eliminated  during  a  period  of  exercise,  as  com- 
pared with  one  of  rest,  may  thus  serve  as  an  index  of  the  amount  of 
muscular  work  done.  I  have  already  pointed  out  that  the  nitro- 
genous constituents  of  the  muscle-tissue  cannot  be  regarded  as  a 
source  of  muscular  energy,  and  that  this  must  be  sought  in  its  non- 
nitrogenous  components.  This  fact  was  well  shown  during  the  ascent 
of  the  Faulhorn  mountain  by  Fick  and  Wislicenus,  in  which  it  was 
calculated  that  the  total  amount  of  work  done  by  the  latter  amounted 
to  at  least  368,000  kilogram  meters.  The  amount  of  nitrogen  which 
he  eliminated  during  the  ascent  and  the  six  hours  following  corre- 
sponded to  37  grammes  of  albumin.  Translated  into  calories,  this 
would  represent  about  106,000  kilogrammeters  of  work.  Deducting 
this  from  368,000,  there  would  remain  262,000  kilogrammeters, 
which  could  not  be  accounted  for  by  a  decomposition  of  nitrogenous 
material,  and  which  must  hence  be  referable  to  the  destruction  in  the 
muscles  of  other  bodies  which  are  free  from  nitrogen.  Of  these,  the 
glycogen  which  is  referable  to  ingested  carbohydrates  is  no  doubt  the 
most  important.  But  while  normally  the  muscle  glycogen  is  prob- 
ably derived  from  this  source  exclusively,  there  is  evidence  to  show 


GLYCOGEN.  355 

that  it  may  be  formed  from  the  albumins  as  well.  If  animals  are 
a  Howe  I  to  starve  until  the  entire  reserve  of  glycogen  has  been  con- 
sumed, and  they  are  then  fed  on  albumins  exclusively,  it  will  be 
observed  that  a  gradual  deposition  of  glycogen  occurs  nevertheless, 
which  can  be  referable  only  to  the  ingested  albumins.  In  the  severer 
forms  of  diabetes,  moreover,  as  will  be  shown  later,  sugar  appears 
in  the  urine  although  all  carbohydrates  are  excluded  from  the 
diet.  Of  the  decomposition-products  of  the  albumins,  however, 
from  which  glycogen  is  formed  synthetically  under  such  conditions, 
we  have  no  knowledge  that  is  definite.  But  it  is  conceivable  that  the 
paralactic  acid,  which  is  now  generally  regarded  as  an  albuminous 
derivative,  and  which,  as  we  shall  presently  see,  is  constantly  formed 
during  the  activity  of  muscle-tissue,  may  here  be  of  moment.  This 
is  a  mere  supposition,  however,  and  lacks  definite  proof. 

Whether  or  not  the  fats  finally  can  also  give  rise  to  the  formation 
of  glycogen  has  not  been  established  beyond  a  doubt.  It  seems, 
however,  that  this  does  not  occur.  At  the  same  time  we  must 
admit  that  there  is  evidence  to  show  that  to  a  certain  extent  they 
can  supply  the  energy  which  is  necessary  for  the  functioning  of 
muscle-tissue  when  a  sufficient  supply  of  glycogen  is  not  avail- 
able. 

While  I  have  stated  above  that  as  a  result  of  muscular  activity 
the  glucose  which  is  derived  from  the  muscle  glycogen  is  decom- 
posed into  carbon  dioxide  and  water,  there  is  evidence  to  show  that 
this  decomposition  does  not  occur  in  the  sense  of  a  direct  oxidation. 
It  is  hence  assumed  that  a  primary  splitting  up  of  the  glucose 
m  tlecule  occurs,  but  of  the  products  which  are  formed  nothing 
definite  is  known.  On  the  one  hand,  we  may  suppose  that  lactic 
acid  thus  results,  but  we  may  also  imagine  that  alcohol  is  produced, 
and  is  then  oxidized  to  carbon  dioxide  and  water.  As  a  matter  of 
fact,  traces  of  alcohol  an;  always  found  when  perfectly  fresh  organs 
are  distilled  with  water  immediately  after  their  removal  from  the 
body. 

Of  the  forces  which  are  at  work  in  effecting  both  the  synthesis 
of  glycogen  and  its  inversion  to  maltose  and  glucose,  and  the  sub- 
8equ  nt  d  •(•  imposition  of  the  latter,  nothing  definite  is  known.  But 
in  view  of  the  constant  presence  of  ptyalin  and  maltase  in  muscle- 
tissue  there  is  some  ground  for  the  assumption  that,  in  part  at  least, 
these  changes  may  be  referable  to  the  action  of  enzymes. 

Isolation. — If  it  is  desired  to  isolate  the  glycogen  from  muscle- 
tissue,  it  i-  necessary  to  place  the  material  in  boiling  water  imme- 
diately after  the  death  of  the  animal,  so  as  to  prevent  its  trans- 
formation into  glucose  and  the  resulting  products  of  decomposition. 
Otherwise  this  will  OCCUr,  as  the  death  of  the  individual  cells  docs 
not  coincide  in  point  of  time  with  (he  death  of  the  animal  as  a 
whole,  ami  there  i-  danger,  moreover,  that  the  inverting  ferments 
of  the  tissue  remain  active.  Thai  this  actually  occurs  can  be 
readily  demonstrated  by  treating  one  portion  of  the  muscle-tissue  as 


356  THE  MUSCLE-TISSUE. 

described,  while  a  second  portion  is  allowed  to  remain  exposed  to 
the  air  for  a  few  hours.  Both  portions  are  then  examined  for 
glycogen,  when  it  will  be  seen  that  only  the  first  gives  a  positive 
reaction.     (As  regards  the  details  of  the  method,  see  page  403.) 

GLUCOSE. 

That  traces  of  glucose  and  maltose  may  be  found  in  fresh  muscle- 
tissue  is,  of  course,  not  surprising  in  view  of  the  above  considerations. 
To  demonstrate  their  presence,  the  fresh  material  is  finely  hashed, 
placed  in  boiling  water,  and  boiled  for  a  few  minutes.  On  cooling, 
the  mixture  is  filtered,  the  filtrate  concentrated  to  a  small  volume 
and  examined  in  the  usual  manner.  Larger  quantities  may  be 
obtained  if  the  finely  minced  tissue  is  placed  in  chloroform-water 
and  autodigestion  is  allowed  to  proceed  for  several  weeks. 

LACTIC  ACID. 

The  reaction  of  living  muscle-tissue  while  at  rest  is  neutral  or 
slightly  alkaline.  After  death,  however,  it  becomes  acid,  and  it 
can  then  be  demonstrated  that  the  acidity  is  in  part,  at  least,  refer- 
able to  paralactic  acid.  Through  the  action  of  the  latter  upon 
dipotassium  phosphate  monopotassium  phosphate  then  results,  and 
a  second  factor  thus  appears,  to  which  the  acid  reaction  is  due. 

Formerly  it  was  supposed  that  rigor  mortis  was  the  result  of  the 
formation  of  lactic  acid,  but  we  now  know  that  this  is  not  the  case, 
and  that  the  coagulation  of  the  muscle-tissue  precedes  the  appearance 
of  the  acid  reaction.  To  use  the  words  of  SalkoAVski,  the  muscle  does 
not  form  lactic  acid  because  it  dies,  but  because  it  lives,  and  only  as 
long  as  it  lives.  With  the  occurrence  of  its  death  the  formation  ceases. 
This  is,  therefore,  a  vital  or  ultravital  process,  and  there  is  abundant 
evidence  to  show  that  this  view,  which  is  now  quite  generally  accepted, 
is  correct.  Under  ordinary  conditions  it  is  difficult  to  show  that 
acid  material  is  produced  while  the  muscle  is  at  work,  as  it  is  then 
removed  by  the  circulation  as  rapidly  as  formed ;  but  if  this  is 
prevented,  the  fact  can  readily  be  demonstrated.  To  this  end,  one 
sciatic  nerve  of  a  rabbit  is  divided  and  the  animal  poisoned  with 
strychnin.  If  then  the  muscles  of  both  legs  are  removed  during  the 
final  convulsions  of  the  animal,  it  will  be  noted  that  the  reaction  of 
those  groups  which  had  remained  in  connection  with  their  nerve- 
supply  is  distinctly  acid,  while  the  others,  the  nerve  of  which  was 
severed,  show  a  neutral  reaction.  That  the  acid  reaction  in  such 
cases  is,  in  part  at  least,  actually  due  to  lactic  acid,  can  be  shown  by 
extracting  the  rested  and  the  tetanized  groups  with  water  and  then 
with  alcohol.  On  evaporating  the  resulting  extracts  and  weighing 
the  corresponding  residue  it  will  be  noted  that  the  weight  of  the 
alcoholic  fraction  is  greater  in  the  case  of  the  worked  muscle  than 


LACTIC  ACID.  357 

of  those  that  have   rested,  while  the   reverse   holds  good   for  the 
aqueous  portions. 

Lactic  acid  is,  however,  produced  by  the  muscle  not  only  when 
at  work,  but  also  while  resting.  This  has  been  shown  by  Zillessen 
and  v.  Frey.  These  observers  found  that  on  transfusing  the  muscles 
of  the  hindquarters  of  a  dog,  during  three  hours,  an  increase  in  the 
amount  of  lactic  acid  resulted,  which,  calculated  for  the  entire  amount 
of  blood,  corresponded  to  as  much  as  1.48  grammes  of  zinc  lactate. 

The  amount  of  lactic  acid  that  may  be  isolated  from  dead  muscles 
while  still  rigid  varies  between  0.1  and  1.0  per  cent.,  and  it  is  note- 
worthy that  for  definite  groups  of  muscles  this  amount  is  constant, 
no  matter  whether  the  formation  of  the  acid  is  allowed  to  proceed 
rapidly  or  slowly.  This,  however,  holds  good  only  for  correspond- 
ing muscles,  and  is  different  in  different  groups.  In  rabbits  larger 
amounts  can  thus  always  be  obtained  from  the  muscles  of  the  trunk 
than  from  those  of  the  extremities. 

To  the  general  rule  that  the  acidity  of  corresponding  muscles  is 
always  the  same,  there  is  one  exception,  viz.,  the  heart,  which  is  the 
only  muscle  of  the  body,  moreover,  that  normally  presents  an  acid 
reaction.  Larger  amounts  of  lactic  acid  are  here  always  found  in 
the  left  than  in  the  right  side. 

As  regards  the  origin  of  lactic  acid  in  muscle-tissue,  it  was  long 
thought  that  the  glycogen  probably  represented  its  principal  source. 
There  area  number  of  facts  indeed  which  favor  such  an  assumption. 
I  have  pointed  out  already  that  after  death  the  glycogen  gradu- 
ally disappears,  and  we  have  just  seen  that  lactic  acid  is  then  found. 
Glycogen  is  similarly  decomposed  during  muscular  activity  in  the 
living  animal,  where  lactic  acid  is  also  constantly  produced,  and,  as 
I  have  shown,  the  same  also  occurs  in  the  muscle  while  at  rest. 
Then  again  there  is  evidence  to  show  that  the  decomposition  of 
glucose  in  the  muscle-tissue  does  not  occur  in  the  sense  of  a  direct 
.oxidation,  but  that  a  primary  division  of  the  molecule  occurs,  and 
that  lactic  acid  may  be  one  of  the  resulting  products.  But,  on  the 
other  hand,  observations  exist  which  go  to  show  that  the  amount  of 
lactic  acid  that  i-  produced  during  rigor  mortis  bears  no  relation  to 
the  amount  of  glycogen  which  was  present  at  the  time,  and  it  has 
further  been  noted  that  lactic  acid  is  still  formed  in  muscles  from 
which  all  glycogen  lias  previously  been  removed  by  starvation.  The 
conclusion  hence  suggests  it-ell'  that  while  ;i  certain  amount  of 
luetic  acid  may  be  derived  from  glycogen,  this  does  not  represent  its 
only  Bource,  and  we  musl  admit  that  to  some  extent  the  albumins 
of  muscle-tissue  also  contribute  toward  its  formation.  There  is  a 
tendency  among  physiological  chemists  at  the  present  time  to  regard 
this  source  indeed  as  the  mosl  important.     This  view  is  largely  based 

upon  observations,  which  go  to  -how  that  increased  amounts  of  lactic 

acid  appear  in  the  urine  whenever  the  formation  of  urea  is  impaired 

in  the  liver,  or  when  this  organ  is  entirely  excluded    from  the  gen- 
eral circulation.     Similar  results  also   have  been  obtained  in  birds, 


358  THE  MUSCLE-TISSUE. 

in  which  uric  acid  represents  the  most  important  end-product  of  the 
normal  nitrogenous  metabolism.  In  geese  it  could  be  demonstrated 
that  after  removal  of  the  liver  the  elimination  of  lactic  acid  was  in 
no  ways  influenced  by  an  increased  or  diminished  ingestion  of  carbo- 
hydrates, while  the  administration  of  larger  amounts  of  albumin 
invariably  results  in  a  corresponding  increase  of  the  lactic  acid. 
When  from  any  reason,  moreover,  albuminous  decomposition  is  in- 
creased, while  the  oxidation-processes  of  the  body  are  at  the  same 
time  diminished,  increased  amounts  of  lactic  acid  are  found  in  both 
the  blood  and  the  urine. 

We  have  seen  that  Siegfried's  phosphor-carnic  acid  gives  rise 
to  the  formation  of  lactic  acid  on  hydrolytic  decomposition,  and  it 
is  thus  possible  that  this  substance  may  be  its  immediate  antecedent. 
Further  researches,  however,  are  necessary  before  the  formation  of 
lactic  acid  from  the  muscle-albumins  can  be  satisfactorily  explained. 
Whether  or  not  enzymatic  influences  are  here  at  work  we  do  not 
know.  Salkowski  denies  this  possibility  on  the  basis  that  lactic  acid 
is  not  found  among  the  products  of  autodigestion  when  perfectly 
fresh  muscle-tissue  is  allowed  to  stand  in  contact  with  chloroform- 
water,  as  the  chloroform,  according  to  this  observer,  does  not  pre- 
vent the  action  of  enzymes.  If  this  property  holds  for  all  ferments, 
the  conclusion  would  also  follow  that  the  formation  of  lactic  acid 
can  neither  be  referable  to  the  action  of  living  protoplasm,  as  the 
chloroform  represents  a  strong  protoplasmatic  poison.  We  have 
seen,  as  a  matter  of  fact,  that  muscle-plasma  also  becomes  acid  after 
the  occurrence  of  coagulation,  and  protoplasmatic  activity  here 
manifestly  does  not  enter  into  consideration.  We  are  hence  forced 
to  the  conclusion  that  the  formation  of  lactic  acid  is  either  referable 
to  the  action  of  a  ferment  which  is  destroyed  by  chloroform,  or  that 
it  results  from  a  spontaneous  decomposition  of  certain  substances 
which  are  especially  unstable. 

Besides  paralactic  acid,  traces  of  common  lactic  acid  also  are  said 
to  occur  in  muscle-tissue.  To  isolate  the  bodies  in  question,  the  fol- 
lowing procedure  may  be  employed. 

Isolation  and  Quantitative  Estimation. — A  carefully  weighed 
amount  of  muscle-tissue  is  finely  hashed,  repeatedly  extracted  with 
cold  water,  and  the  mixture  passed  through  a  muslin  filter.  The 
resulting  fluid  is  feebly  acidified  with  sulphuric  acid  and  boiled, 
so  as  to  remove  the  coagulable  albumins.  Baryta-water  is  now 
added  so  long  as  a  precipitate  is  formed  ;  this  is  filtered  off.  The 
filtrate  is  freed  from  the  barium  that  was  added  in  excess  by  pass- 
ing carbon  dioxide  into  the  solution,  when  it  is  boiled,  filtered,  and 
concentrated  to  a  thin  syrup.  Care  should  be  taken,  however,  that 
the  temperature  does  not  exceed  70°  C.  toward  the  end.  The  re- 
sulting material  is  treated  with  ten  times  its  volume  of  absolute 
alcohol,  set  aside  for  a  while,  and  filtered.  The  alcoholic  solution  is 
evaporated  on  a  water-bath,  and  the  remaining  thick  syrup  treated 
with  about  an  equal  amount  of  a  moderately  dilute  solution  of  phos- 


IXOSIT.  359 

phoric  acid.  This  liberates  the  lactic  acid  from  its  salts,  while  the 
chlorides  and  sulphates  remain  unaffected.  The  lactic  acid  is  then 
extracted  with  ether.  The  ether  is  distilled  off,  the  residue  boiled 
with  water  and  an  excess  of  carbonate  of  zinc,  and  filtered  while  still 
hot.  The  filtrate  is  concentrated  to  a  small  volume,  when  on  stand- 
ing, and  especially  after  the  addition  of  a  small  amount  of  alcohol, 
the  zinc  lactate  crystallizes  out.  To  separate  the  paralactate  from 
the  common  lactate,  which,  as  I  have  said,  is  also  found  in  traces  in 
the  muscle-tissue,  the  crystals  are  placed  in  absolute  alcohol,  which 
dissolves  the  paralactate  (solubility  1  :  1100),  while  the  common  form 
is  insoluble.     They  are  then  finally  dried  and  weighed. 

To  obtain  the  lactic  acid  as  such  the  lactates  are  decomposed  with 
hydrogen  sulphide.  The  resulting  zinc  sulphide  is  filtered  off, 
washed  with  water,  when  filtrate  and  washings  are  evaporated  at 
70°  C.  to  a  small  volume. 

Both  acids  are  amorphous,  and  are  obtained  in  the  form  of  a  thick 
syrup,  which  is  soluble  in  water,  alcohol,  and  ether.  Of  their  salts, 
tiie  zinc  salts  are  especially  characteristic  and  serve  to  distinguish 
the  two  forms  from  each  other.  As  I  have  indicated,  the  common 
lactate  is  insoluble  in  absolute  alcohol.  It  crystallizes  with  three 
molecules  of  water,  which  escape  at  a  temperature  of  105°  C,  so 
that  the  loss  of  weight  will  then  correspond  to  18.18  per  cent.  The 
paralactate,  on  the  other  hand,  is  soluble  in  absolute  alcohol,  though 
with  difficulty,  and  crystallizes  out  with  only  two  molecules  of  water, 
which  likewise  escapes  at  105°  C.  In  this  case  the  loss  of  weight 
amounts  to  12  per  cent.  Both  the  free  acid  and  the  paralactate  are 
lsevorotatory,  while  the  common  form  and  its  salts  are  optically 
inactive. 

INOSIT. 

Of  the  origin  of  inosit,  which  is  apparently  a  constant  constituent 
of  muscle-tissue,  but  which  is  found  also  in  other  organs  of  the 
body,  and  appears  in  the  urine  when  polyuria  is  either  artificially 
produced  or  results  from  some  morbid  process,  nothing  is  known. 
It  is  not  peculiar  to  the  animal  world,  however,  but  occurs  widely 
distributed  in  the  vegetable  kingdom  also,  and  is  identical  with  the 
90-called  phaseo-mannite,  which  is  especially  abundant  in  certain 
In  in-.      In   muscle-tissue   it   is   found   only   in    traces. 

The  substance  is  not  a  carbohydrate,  as  was  once  supposed,  but 
belongs  to  the  aromatic   series, and   is  commonly  regarded  as  hexa- 

hydroxybenzol : 

CH.OH 

on. in       <  ji  on 

I 
oil. lie        CH.OB 


<  n.on 


In  pure  form  it  crystallizes  in  colorless,  monoclinic  prisms,  which 
are  often   grouped   in   rosettes.     Ii   melts  at   217°  C.     [t  is  soluble 


360  THE  MUSCLE-TISSUE. 

in  water  and  dilute  alcohol,  but  is  insoluble  in  absolute  alcohol  and 
ether.  The  substance  does  not  reduce  the  metallic  oxides  in  alka- 
line solution,  and  is  optically  inactive.  It  is  not  fermentable  with 
common  yeast,  but  is  decomposed  by  the  Bacterium  lactis  with  the 
formation  of  lactic  acid,  and  subsequently  yields  butyric  acid. 

Tests. — Scherer's  Test. — If  a  few  crystals  of  inosit  are  evaporated 
on  platinum  foil  with  a  little  nitric  acid,  and  the  residue  is  treated 
with  ammonia  and  a  drop  of  dilute  solution  of  calcium  chloride,  a 
rose-red  spot  remains  on  further  evaporation.  The  reaction  is  due 
to  the  formation  of  rhbdizonic  acid. 

Gallois'  Test. — On  evaporating  a  small  amount  of  a  solution  of 
inosit  and  adding  a  few  drops  of  a  dilute  solution  of  mercuric 
nitrate  before  the  residue  has  become  dry,  a  yellow  spot  develops 
on  the  further  application  of  heat,  which  ultimately  turns  red.  On 
cooling,  the  red  color  disappears,  but  reappears  on  heating. 

Isolation. — To  demonstrate  the  presence  of  inosit  in  muscle- 
tissue,  this  is  finely  hashed,  and  extracted  with  hot  water,  when  the 
albumins  are  removed  by  boiling.  The  filtrate  is  precipitated  with 
barium  hydrate,  so  as  to  remove  the  phosphates  that  are  present. 
After  filtering  the  liquid  is  then  concentrated,  until  most  of  the 
kreatin  has  separated  out.  This  is  removed  by  filtration  ;  the  fil- 
trate is  boiled  with  four  times  its  volume  of  alcohol ;  the  solution 
is  allowed  to  cool,  freed  from  the  mineral  constituents  that  have 
separated  out,  and  shaken  with  ether.  The  inosit  then  separates 
out  in  the  form  of  fine  platelets,  which  can  be  further  purified  by 
dissolution  in  alcohol  and  reprecipitation  with  ether. 

The  scyllite  which  is  found  in  cartilaginous  fish,  where  inosit 
is  absent,  is  closely  related  to  the  latter,  and,  like  this,  gives  the 
reaction  of  Gallois. 

THE   NITROGENOUS   EXTRACTIVES. 

The  nitrogenous  extractives  of  muscle-tissue  comprise  the  com- 
mon derivatives  of  the  nuclear  nucleins,  viz.,  xanthin,  hypoxanthin, 
guanin,  and  carnin ;  further,  traces  of  taurin,  glycocoll,  urea,  and 
uric  acid ;  and  more  abundantly  kreatin  and  kreatinin. 

Kreatin  and  Kreatinin. — Kreatin  is  a  constant  constituent  of 
the  muscle-tissue  of  the  vertebrate  animals,  while  in  the  invertebrates 
it  has  not  as  yet  been  found.  Its  amount  is  often  quite  considera- 
ble, and  it  has  been  calculated  that  in  adult  man  as  much  as  90 
grammes  could  be  extracted  from  the  muscles  of  the  entire  body. 
Its  anhydride,  kreatinin,  on  the  other  hand,  is  usually  found  only 
in  traces,  but  may  occur  in  larger  amounts,  and  notably  in  certain 
fishes. 

Of  the  origin  of  kreatin  little  is  known,  and  it  is  noteworthy 
that  the  substance  has  thus  far  not  been  obtained  from  the  animal 
tissues  directly  by  artificial  means.  It  has  been  found  in  the  brain, 
in  the  thyroid  gland,  in  the  blood,  in  transudates,  in  the  amniotic 


THE  NITROGENOUS  EXTRACTIVES.  361 

fluid,  and  is  also  a  constant  constituent  of  the  urine:     In  the  vege- 
table world  it  does  not  occur. 

From  the  observation  of  St.  Johnson  that  the  urinary  kreatinin 
is  not  identical  with  the  substance,  which  can  be  isolated  from 
muscle-tissue,  it  has  been  concluded  that  the  former  may  not  be 
derived  from  the  muscles  at  all,  but  may  possibly  be  referable  to  the 
kreatin,  which  is  found  in  other  organs  of  the  body  and  notably  in 
the  thyroid  gland.  Its  identity  with  these  kreatinius,  however,  has 
not  vet  been  established,  nor  is  there  reason  to  suppose  that  these 
forms  differ  from  the  common  kreatinin  of  the  muscles.  However 
this  may  lie,  the  kreatins,  viz.,  kreatinius,  are  essentially  specific 
decomposition-products  of  muscle-tissue,  and  are  unquestionably 
derived  from  the  common  muscle-albumins.  This  is  suggested  by 
the  observation  that  larger  amounts  of  kreatin  and  kreatinin  can  be 
isolated  from  muscles  that  have  previously  been  worked,  than  from 
muscles  that  have  been  at  rest.  I  have  shown,  it  is  true,  that  the 
nitrogenous  components  of  the  muscle-tissue  enter  into  considera- 
tion only  in  a  secondary  manner,  as  a  source  of  muscular  energy, 
but  this  does  not  preclude  the  possibility,  that  during  work  the 
metabolism  of  the  muscle-albumins  is  increased.  That  such  an 
increase  will  of  necessity  become  more  apparent  during  starvation  is, 
of  course,  -elf-evident,  and  we  find,  as  a  matter  of  fact,  that  under 
such  conditions  a  corresponding  increase  in  the  formation  of  kreatin 

occurs. 

While  our  knowledge  of  the  manner  in  which  kreatin  and 
kreatinin  are  produced  in  the  body  is  as  yet  practically  nil,  we  know, 
on  the  other  hand,  that,  when  once  formed,  it  is  further  decomposed, 
and  contributes  toward  the  formation  of  urea.  Artificially  this  can 
readily  lie  accomplished  by  boiling  kreatin  with  baryta-water,  when 
it  is  decomposed  with  the  formation  of  urea  and  mcthyl-glycocoll. 
At  the  game  time,  however,  methyl-hydantoin  and  ammonia  result. 
Whether  or  not  the  decomposition  of  kreatin,  which  is  now  known 
to  occur  in  the  muscle-tissue  itself,  takes  place  in  the  same  manner, 
i-  not  known.  But  if  so,  we  could  readily  understand  why  traces 
of  glycocoll  are  also  so  constantly  met  with.  Both  substances,  how- 
ever, are  manifestly  removed  as  rapidly  a-  possible,  as  neither  urea 
nor  glycocoll  is  ever  found  in  the  tissue  itself  beyond  traces.1 

In  this  connection  it  is  interesting  to  note  that  Guareschi  and 
Mosso  have  succeeded  in  extracting  methyl-hydantoin  also  from  the 
mil-el,-  of  the  calf.      Thai  methyl-hydantoin  belongs  to  the  class  of 

ureids  has  already  been  pointed  out,  and  we  may  therefore  assume 
thai   the  substance  is  further  decomposed  with   the    formation  of 

urea,  if  further  researches  should  -how  that  the  decomposition  of 
kreatin   actually   takes     place   in    the   living  tissue  also,  as  outlined 

above.     The  -mail   amount  of  ammonia,  which   at  the  same  time 

1  Tlii-  statement  requires  modification,  ne  quite  considerable  amounts  of  urea 
can  be  demonstrated  in  the  muscles  of  certain  fishes,  such  us  the  Bhark  and  the 
;  eon, 


362  THE  MUSCLE-TISSUE. 

results,  possibly  combines  with  lactic  acid,  and  is  then  further  trans- 
formed into  urea  in  the  liver. 

While  the  kreatin,  which  is  thus  produced  during  the  nitrogenous 
metabolism  of  the  muscle-tissue,  is  largely  transformed  into  urea,  it 
is  noteworthy  that  the  substance,  when  ingested  by  the  mouth, 
reappears  in  the  urine  in  practically  the  same  amount.  This  ob- 
servation is  explained  by  the  assumption  that  the  kreatin  in  this 
case  does  not  pass  through  the  muscles,  and  thus  escapes  decom- 
position, and  there  can  be  no  doubt  that  a  certain  fraction  of  the 
kreatin  which  is  eliminated  in  the  urine  is  referable  to  this  source. 

Properties. —  Kreatin  crystallizes  in  rhombic  prisms,  with  one 
molecule  of  water,  which  escapes  at  100°  C.  It  is  readily  soluble 
in  warm  water,  less  so  in  cold  water,  and  is  insoluble  in  alcohol  and 
ether. 

As  has  been  indicated  before,  it  can  be  formed  synthetically  from, 
cyanamide  and  methyl-glycocoll,  according  to  the  equation  : 

CN.NH2   +   CH2.NH(CH3).COOH    =   NH=C< 

Cyanamide.  Methyl-glycocoll.  \Nt(CH3).CH2.COOH 

Kreatin. 

On  boiling  its  acidified  aqueous  solutions,  the  substance  loses 
water  and  is  transformed  into  kreatinin,  from  which  the  kreatin  is 
again  obtained  by  treating  with  dilute  alkaline  solutions.  The 
transformation  of  kreatinin  into  kreatin  can  indeed  take  place  in  the 
aqueous  solution  directly,  and  may  be  hastened  by  the  application 
of  heat.  The  relation  between  the  two  substances  can  thus  be 
illustrated  by  the  equation  : 

/NH2  /NH 

NH=C<  =  NH=C<  +  H20 

\N(CH3).CH2.COOH  \N(CHs).CH2.CO 

Kreatin.  Kreatinin. 

The  same  relation  thus  exist?  between  kreatin  and  kreatinin  as 
between  glucocyamin  and  glucocyamidin,  and,  as  a  matter  of  fact, 
both  are  methyl-substitution-products  of  the  two  latter,  and  are 
accordingly  also  termed  methyl-glucocyamin  and  methyl-glucocy- 
amidin. 

/NH,  /NH.2 

NH=C<  NH=C< 

\NH.CH2.COOH  \N(CH3).CH2.COOH 

Glucocyamin.  Kreatin. 

.NH— CO  /NH^ 

NH=C<  |  NH=C,  ^^^ 

\nh.   CH2.  \N(CH3).CH,CO 

Glucocyamidin.  Kreatinin. 

Kreatinin  crystallizes  in  prisms,  without  water  of  crystallization, 
and  is  soluble  in  water  and  alcohol  (see  also  pages  84  and  246). 

Isolation  of  Kreatin. — To  isolate  kreatin  from  muscle-tissue,  this 
is  finely  hashed  and  repeatedly  extracted  with  an  equal  weight  of 
water  at  a.  temperature  of  from  55°  to  60°  C.     The  extracts  are 


THE  XANTHIN-BASES.  363 

boiled  so  as  to  remove  coagulable  albumins.  The  filtrate  is  pre- 
cipitated with  subacetate  of  lead,  care  being  taken  to  avoid  an 
excess.  The  resulting  filtrate  is  freed  from  lead  by  hydrogen  sul- 
phide, and  is  concentrated  to  a  small  volume  at  as  low  a  temperature 
as  possible.  On  standing,  the  kreatin  crystallizes  out,  and  can  then 
be  purified  as  desired.  To  identity  the  substance,  it  is  conveniently 
transformed  into  kreatinin  by  prolonged  boiling  (one-half  to  one 
hour)  with  dilute  hydrochloric  acid.  The  resulting  material  is  then 
examined  as  described   in  the  section  on  the  Urine  (page  240). 

In  addition  to  the  common  kreatins  which  have  just  been  con- 
sidered, Gautier  succeeded  in  further  isolating  xanthokreatinin, 
crusokreatinin  and  amphykreatin  from  muscle-tissue.  The  sub- 
stances are,  however,  found  only  in  traces  and  have  not  as  yet  been 
studied  in  detail. 

THE  XANTHIN-BASES. 

The  xanthin-bases  which  have  been  isolated  from  muscle-tissue 
comprise  xanthin,  hypoxanthin,  and  guanin.  In  addition,  an- 
other basic  substance  has  been  obtained,  which  is  termed  carnin. 
This  is  manifestly  closely  related  to  the  xan thins  proper,  as  on 
oxidation  it  is  transformed  into  hypoxanthin.  These  bodies  are 
formed  during  the  metabolism  of  the  muscle  nuclei,  and  are  in  part 
eliminated  in  the  urine  as  such.  A  variable  fraction,  however,  is 
directly  oxidized  to  uric  acid,  which  in  turn  may  contribute  to  the 
formation  of  urea,  but  is  in  part  also  eliminated  directly  (see  pages 
222  and'233). 

Isolation. — To  demonstrate  the  presence  of  the  xanthin  bases  in 
muscle-tissue  it  is  necessary  to  work  with  large  amounts  of  material, 
as  the  quantity  present  is  always  small.  On  the  whole  it  is  more 
convenient  to  start  with  some  extract  of  beef,  such  as  that  of  Liebig, 
and  to  proceed  as  follows: 

The  material  in  question  is  taken  up  with  an  amount  of  water 
which  is  just  sufficient  for  its  solution,  at  a  temperature  of  about 
50  C.  The  liquid  is  then  precipitated  with  a  solution  of  sub- 
acetate  of  lead.  The  resulting  filtrate  we  term  A.  The  pre- 
cipitate contains  the  carnin  in  combination  with  lead.  To  isolate 
the  substance  the  mass  is  suspended  in  water  and  boiled.  The 
filtrate  while  still  hot  is  saturated  with  hydrogen  sulphide,  which 
decomposes  the  lead  compound  of  carnin  with  the  liberation  of  the 
free  base.  The  lead  sulphide  is  filtered  off,  the  filtrate  concen- 
trated to  a  small  volume  and  precipitated  with  a  concentrated 
solution  of  silver  nitrate.  Ammonia  is  added  to  dissolve  any  pre- 
cipitated chlorides  when  the  insoluble  silver-carnin  is  placed  in 
a  small  amount  of  hot  water  and  is  decomposed  with  hydrogen 
sulphide.  The  -<,luti<,ii  i-  filtered  while  hot,  decolorized  with 
animal  charcoal, and  precipitated  with  alcohol.  The  carnin  is  thus 
thrown  down. 


364  THE  MUSCLE-TISSUE. 

Filtrate  A  is  decomposed  with  hydrogen  sulphide,  filtered  and 
strongly  concentrated,  when  on  standing  kreatin  separates  out. 
The  filtrate  is  rendered  alkaline  with  ammonia  and  is  precipitated 
with  ammoniacal  silver  nitrate  solution.  The  xanthin  bases  are  thus 
thrown  down  as  double  silver  salts.  They  are  filtered  off  and  dis- 
solved in  a  small  amount  of  hot  nitric  acid.  The  solution  is  placed 
in  the  refrigerator  when  the  salts  of  hypoxanthin  and  guanin  crystal- 
lize out.     The  filtrate  is  termed  B. 

To  separate  the  guanin  from  the  hypoxanthin,  the  material  is  sus- 
pended in  water ;  the  liquid  is  brought  to  the  boiling-point  and 
treated  with  a  solution  of  ammonium  sulphide,  drop  by  drop.  The 
silver  salts  are  thus  decomposed.  The  liquid  is  filtered  while 
hot.  The  filtrate  C  contains  a  portion  of  the  guanin  and  all  of 
the  hypoxanthin,  while  a  fraction  of  the  guanin  remains  in  the 
precipitate.  This  can  be  extracted  by  boiling  with  a  very  dilute 
solution  of  hydrochloric  acid,  when  the  free  base  is  precipitated 
by  adding  an  excess  of  ammonia  to  the  acid  solution.  Filtrate  C 
is  placed  on  a  water-bath  and  treated  with  ammonia,  when  the 
remaining  portion  of  the  guanin  is  thrown  down.  The  hypoxanthin 
is  then  obtained  after  filtration  on  evaporating  the.  ammoniacal 
solution. 

Filtrate  B  contains  the  xanthin  salt  of  silver  nitrate.  To  isolate 
the  free  base  the  double  salt  is  first  precipitated  from  its  acid  solu- 
tion by  ammonia.  It  is  suspended  in  water,  decomposed  with 
hydrogen  sulphide,  and  extracted  with  ammonia.  On  evaporation 
the  xanthin  crystallizes  out. 

This  method  is  well  adapted  for  extracting  the  xanthin  bases 
from  any  organs  of  the  body,  and  also  serves  for  the  isolation  of 
adenin.  Should  this  be  present,  it  is  found  in  filtrate  C.  On  adding 
ammonia  the  adenin  together  with  the  hypoxanthin  remains  in  solu- 
tion. On  cooling  the  adenin  separates  out,  especially  after  the 
ammonia  has  been  evaporated  off.  Hypoxanthin  remains  in  solu- 
tion and  is  obtained  on  final  evaporation. 

As  the  general  chemical  relations  of  the  xanthin-bases  have 
already  been  considered  (page  77),  it  will  suffice  to  give  a  brief 
account  of  the  more  important  properties  of  the  individual  sub- 
stances at  this  place. 

Xanthin. — Pure  xanthin  is  either  amorphous  or  occurs  in  the  form 
of  fine  platelets  which  are  gathered  into  little  clumps  so  that  the 
material  often  presents  a  granular  aspect.  In  cold  water  it  is  almost 
insoluble,  and  in  hot  water  also  it  dissolves  with  difficulty.  In 
alcohol  and  ether  it  is  insoluble.  In  acids  and  alkalies  it  dissolves 
with  comparative  ease,  but  at  the  same  time  it  combines  with  these 
to  form  compounds,  which  are  for  the  most  part  readily  crystalliz- 
able.  From  its  ammoniacal  solution  it  is  obtained  as  such  on  evapo- 
ration of  the  ammonia.  From  this  solution  it  is  thrown  down 
by  silver  nitrate  as  a  gelatinous  precipitate  of  the  composition 
C-H4N402.Ag?0.      If  this  is  dissolved  in  nitric  acid,  a  double  salt 


THE  XANTHIN-BASES.  365 

results,  which  crystallizes  out  on  standing.  Unlike  hypoxanthin, 
xanthin  is  precipitated  by  an  ammoniacal  solution  of  lead  sub- 
acetate.  From  its  aqueous  solution  it  is  precipitated  as  a  greenish- 
yellow  material  by  means  of  cupric  acetate  on  boiling. 

Tests. — Nitric  Acid  Test. — On  evaporating  a  few  crystals  of 
xanthin  on  platinum  foil  with  nitric  acid  a  yellow  spot  remains, 
which  turns  red  when  moistened  with  a  drop  of  sodium  hydrate 
solution.  On  further  heating,  it  becames  a  beautiful  purplish  violet. 
The  reaction  is  thus  similar  to  the  murexid  test  for  uric  acid,  but  it 
will  be  noted  that  in  this  case  a  red  color  develops  on  the  addition 
of  the  alkali,  while  with  uric  acid  a  blue  color  is  obtained. 

HorPE-SEYLER's  Test.— A  few  crystals  of  xanthin  are  placed 
in  a  watch-crystal  containing  a  mixture  of  a  few  drops  of  a  solution 
of  sodium  hydrate  and  of  calcium  hypochlorite.  A  dark-green  zone 
then  appears  about  the  xanthin,  which  subsequently  turns  brown 
and  ultimately  disappears. 

Weidel's  Test. — A  small  amount  of  xanthin  is  covered  with 
freshly  prepared  chlorine-water  containing  a  trace  of  nitric  acid.  The 
mixture  is  evaporated  to  dryness  on  a  water-bath,  and  the  residue 
exposed  to  the  fumes  of  ammonia,  when  a  beautiful  red  or  purplish- 
violet  color  develops. 

Hypoxanthin  (Sarcin). — Hypoxanthin  crystallizes  in  small  white 
needles.  It  is  soluble  in  hot  water,  less  readily  so  in  cold  water, 
while  in  cold  alcohol  it  is  almost  insoluble.  Like  xanthin,  it  dis- 
solves with  comparative  ease  in  dilute  solutions  of  the  alkaline 
hydrates  and  acids,  forming  salts,  which  are  decomposed  by  distilled 
water,  with  the  liberation  of  the  free  base.  Of  these,  the  chlorhydrate 
is  fairly  characteristic,  as  on  rapid  evaporation  it  crystallizes  out  in 
distinct  whetstone  crystals  similar  to  those  of  uric  acid.  On  treat- 
ing the  substance  in  ammoniacal  solution  with  an  ammoniacal 
-solution  of  silver  nitrate,  a  double  salt  of  hypoxanthin  with  silver 
i-  precipitated,  which  is  soluble  with  difficulty  in  boiling  nitric  acid. 
(),  cooling,  it  separates  out  in  the  form  of  curiously  bent  prisms, 
which  are  quite  characteristic.  On  boiling  with  a  solution  of  acetate 
of  copper,  hypoxanthin  is  thrown  down  as  a  cupric  salt. 

Test. — The  substance  does  not  give  the  common  reactions  of 
xanthin.  When  treated  with  zinc  and  hydrochloric  acid,  however, 
hypoxanthin  gives  rise  to  a  ruby-red  color,  which  later  changes  to  a 
brownish  redj  when  -odium  hydrate  solution  is  added  in  excess. 

Guanin. — Guanin  is  usually  obtained  in  amorphous  form, but  can 
be  brought  to  crystallize  out  from  its  solutions  in  strong  ammonia, 
on  spontaneous  evaporation.  It  is  insoluble  in  water,  alcohol,  and 
ether,     iii   mineral  acids  it  dissolves  with   comparative  ease,  at  the 

same  time    forming    salt-like  products,  which    are    cryslallizable,  but 

quite  unstable,  ao  that  in  the  case  .,1'  some  of  them  nt  least  the  free 
base  i-  liberated  by  water.  With  the  common  alkalies  it  likewise 
combines  to  form  compounds  which  are  somewhat  soluble  in  warm 
water,  but   more  readily  so  if  a  little  fixed  alkali  is  present,     in 


366  THE  MUSCLE-TISSUE. 

ammonia  it  dissolves  with  great  difficulty,  so  that  it  is  possible  to 
precipitate  the  substance  from  its  acid  solutions  by  the  addition  of 
ammonia.  Silver  nitrate  precipitates  the  substance  from  its  solu- 
tions in  nitric  acid  as  a  double  salt,  which  dissolves  in  boiling  nitric 
acid,  and  crystallizes  out  on  cooling  in  the  form  of  fine  needles.  On 
boiling  a  solution  of  guanin  (as  calcium  compound)  with  acetate  of 
copper  the  corresponding  salt  is  thrown  down. 

Tests. — Like  xanthin,  guanin  gives  the  nitric  acid  reaction,  but 
with  a  somewhat  more  bluish- violet  color,  while  Hoppe-Seyler's  test 
and  that  of  AVeidel  are  negative.  With  picric  acid  it  combines  to 
form  a  yellow  crystalline  precipitate  when  a  saturated  solution  of 
the  acid  is  added  to  a  wTarm  solution  of  the  hydrochlorate. 

Adenin. — Adenin  crystallizes  with  three  molecules  of  water  in  the 
form  of  long  hexagonal  needles.  If  these  are  placed  in  an  amount 
of  water  which  is  insufficient  for  their  solution  and  heat  is  now 
applied  to  the  temperature  of  53°  C,  they  suddenly  lose  their  trans- 
parency and  become  opaque.  This  peculiar  behavior  may  aid  in  the 
identification  of  the  substance.  It  is  soluble  with  difficulty  in  cold 
water,  more  readily  so  in  hot  water,  but  is  insoluble  in  ether.  In 
hot  alcohol  it  dissolves  to  a  slight  extent.  In  acids  and  alkalies  it 
is  soluble  with  ease.  In  ammonia  it  is  less  readily  soluble  than 
hypoxanthin,  but  more  readily  so  than  guanin.  From  its  alkaline 
solutions  the  free  base  is  precipitated  by  the  addition  of  very  dilute 
acids,  care  being  taken  to  avoid  an  excess.  With  silver  nitrate  it 
forms  a  compound  which  is  soluble  with  difficulty  in  boiling  nitric 
acid,  and  crystallizes  out  on  cooling.  Like  the  xanthin  bases,  that 
have  already  been  considered,  it  is  precipitated  from  its  solutions  on 
boiling  wTith  acetate  of  copper  as  a  double  salt. 

Tests. — Like  hypoxanthin,  it  does  not  give  the  common  xanthin 
reactions,  nor  does  it  react  with  zinc  and  hydrochloric  acid.  It  is 
most  readily  identified  by  the  behavior  of  its  crystals,  as  has  just 
been  described,  or  by  adding  a  solution  of  auric  chloride  to  its 
solution  as  a  hydrochlorate,  when  a  double  salt  is  formed,  which 
crystallizes  in  part  in  octahedral  or  prismatic  crystals,  the  angles  of 
which  are  often  rounded  off. 

Carnin. — Carnin,  as  I  have  stated  before,  is  not  a  true  xanthin- 
base,  but  is  manifestly  closely  related  to  this  group,  as  on  oxidation 
with  bromine-water  or  with  nitric  acid  it  is  transformed  into  hypo- 
xanthin. Its  formula  and  transformation  into  hypoxanthin  are  seen 
in  the  equation  : 

C7H8N403     +     2Br     =     C5H4N,O.HBr     +     CH3Br    +    C02 
Carnin.  Hypoxanthin-brom-  Methyl- 

hydrate,  bromide. 

Its  structural  formula  in  its  relation  to  hypoxanthin  is  seen  below : 


CH3.N CO  HN CO 

CHOH     C NET  HC        C NHX 

I                              ^CH  ||         I              >CH 

HN— CO — CO N^  N C N^ 


GASES.  367 

It  is  obtained  in  indistinctly  crystalline  form.  In  hot  water  it  is 
readily  soluble,  while  in  cold  water  it  dissolves  with  great  difficulty, 
and  is  insoluble  in  alcohol  and  ether.  It  is  neutral  in  reaction,  and 
combines  with  acids  and  alkalies  to  form  salts.  These  salts  are  not 
decomposed  by  water.  Its  hydrochlorate,  which  results  when  adenin 
is  dissolved  in  warm  hydrochloric  acid,  crystallizes  out  on  cooling, 
and  combines  with  platinum  chloride  to  form  a  double  salt.  Silver 
nitrate  precipitates  it  from  its  aqueous  solutions  as  a  silver  salt,  and 
it  is  to  be  noted  that  this  compound  is  insoluble  both  in  ammonia 
and  nitric  acid.  Subacetate  of  lead  precipitates  adenin  as  a  lead 
salt ;  this  is  soluble  in  boiling  water.  Acetate  of  copper  produces 
no  precipitate.  The  substance  gives  none  of  the  common  reactions 
of  the  xanthin -bases,  and  is  best  identified  by  the  behavior  of  its 
lead  and  silver  salts. 

Still  other  nitrogenous  extractives  may  be  obtained  from  muscle- 
tissue,  but,  with  the  exception  of  taurin  and  inosinic  acid,  they  are 
scarcely  known.     For  a  description  of  taurin  see  page  153. 

Inosinic  Acid. — Inosinic  acid  is  apparently  a  constant  con- 
stituent of  muscle-tissue,  but  is  most  abundantly  encountered  in  the 
muscles  of  ducks,  from  which  Creite  was  able  to  isolate  as  much  as 
0.2b'  per  cent.,  calculated  as  barium  salt. 

The  substance  has  the  composition  CU)H13X4POs,  and  is  com- 
monly regarded  as  a  nucleinic  acid.  On  decomposition  with  boiling 
water  it  is  -aid  to  yield  hypoxanthin,  trioxy-valerianic  acid,  and 
phosphoric  acid.  Whether  or  not  a  relationship  exists  between 
inosinic  acid  and  phosphor-carnic  acid  is  as  yet  unknown. 

GASES. 

Both,  when  at  rest  as  also  during  its  activity,  the  muscle-tissue  is 
constantly  taking  up  oxygen  from  the  blood  and  the  lymph.  This 
\-  stored  in  the  cells  proper,  and  is  extensively  utilized  in  the  oxida- 
tion-processea  which  are  constantly  going  on,  but  which  occur  with 
increased  intensity  when  the  muscle  is  at  work.  Carbon  dioxide  is 
similarly  given  oil*,  and  it  can  be  readily  proved  that  the  oxygen 
which  is  utilized  in  its  formation  is  in  part  at  least  stored  within  the 
tissue.  Carbon  dioxide  is  thus  -till  given  off,  even  when  a  muscle 
i-  removed  from  the  body  and  worked  in  an  atmosphere  which  is 
free  from  oxygen.  It  has  been  noted,  moreover,  that  the  amount 
which  is  then  gel  i'vr  \<  the  same  as  that  which  results  when  the 
muscle  is  worked  in  the  presence  of  an  abundance  of  oxygen.  Of 
the  form,  however,  in  which  the  gas  exists  in  the  muscle  we  know 
nothing,  bul   it   is  manifestly  not    present   in  the  free  state,  as  no 

Oxygen  at  all.  or  very  -mall  amounts  only,  can  be  extracted  by  a 
vacuum  pump.  With  increasing  activity  larger  amounts  of  oxygen 
■'ire    taken    up,    while    larger   amounts   of    carbon   dioxide  are   being 

given  off.     This  difference  is  well  shown  in  the  following  table, 


368  THE  MUSCLE-TISSUE. 

which  is  taken   from  Gautier.     The  figures  have  reference  to  100 
volumes  of  blood,  calculated  at  0°  C.  and  1000  Hgmm.  pressure: 

Carbon 
Oxygen,      dioxide. 

Arterial  blood  from  muscle-tissue 15.25  26.71 

Venous  blood  from  muscle-tissue  while  at  rest  .    .       6.70  33.20 

Venous  blood  from  muscle-tissue  while  at  work    .    .       2.97  36.38 

In  addition  to  carbonic  acid,  small  amounts  of  nitrogen  can  fur- 
ther be  obtained  from  muscle-tissue,  which  are  manifestly  absorbed 
from  the  blood  and  apparently  exist  in  a  state  of  solution.  As  in 
other  tissues  and  fluids  of  the  body  where  nitrogen  is  also  found,  its 
presence  is  probably  of  no  significance.  The  quantity  that  can  be 
obtained  by  the  vacuum  pump  is  essentially  the  same  as  that  which 
is  found  in  the  lymph  and  in  the  blood. 

FAT. 

The  amount  of  fat  which  is  found  in  muscle-tissue  varies  not  only 
with  different  animals  but  also  in  one  and  the  same  individual  at 
different  periods  of  life.  Some  of  the  analytical  results  which  have 
been  obtained  are  shown  below  : 

Pro  mille. 

Lean  beef 6.1-7.6 

Eabbit       10.7 

Partridge 14.3 

Pig 40.0-90.0 

Salmon      100.0 

Mackerel      ■ 164.0 

Eel 329.0 

The  fat  is  deposited  not  only  in  the  interfibrillary  connective 
tissue,  but  also  in  the  sarcoplasm  proper,  and  is  apparently  more 
abundant  in  the  red  meat,  which  contains  more  sarcoplasm  than  in 
white  meat. 

Like  glycogen,  it  here  represents  a  reserve  source  of  muscular 
energy,  but  is  apparently  utilized  more  especially  when  a  sufficient 
supply  of  the  former  or  of  grape-sugar,  as  such,  is  not  available. 

While  it  is  ordinarily  derived  from  the  ingested  fats  or  from  carbo- 
hydrates, there  can  be  no  doubt  that  under  certain  pathological  condi- 
tions, which  are  associated  with  an  increased  destruction  of  tissue 
albumins,  it  can  also' originate  from  these.  This  question,  however, 
we  shall  not  consider  in  detail  at  this  place,  but  shall  revert  to  it  in 
a  future  section. 

In  addition  to  fats,  muscle-tissue  also  contains  a  small  amount  of 
cholesterin,  fats,  and  fatty  acids,  and  at  times  considerable  quanti- 
ties of  lecithins  (0.69  per  cent.). 

The  chemical  composition  of  involuntary  muscle-tissue  is  essen- 
tially the  same  as  that  of  the  striped  variety. 


CHAPTER   XVII. 


THE  NERVE-TISSUE. 


Owixg  to  the  difficulty  which  attends  the  separation  of  the  vari- 
ous morphological  components  of  nerve-tissue,  and  the  peculiar  prop- 
erties of  some  of  its  most  important  chemical  constituents,  it  is  as 
yet  impossible  to  give  an  account  of  its  chemical  composition  which 
is  at  all  satisfactory.  Here,  as  in  the  other  organs  of  the  body,  we 
meet  with  certain  substances  which  are  generally  spoken  of  as  ex- 
tractives, and  which  are  manifestly  katabolic  products  that  result 
during  the  functional  activity  of  the  tissue  in  question.  These  are 
in  no  sense  specific  of  nerve-tissue,  however.  They  comprise  kreatin, 
uric  acid,  urea,  xanthin,  hypoxanthin,  guanin,  adenin,  inosit,  and 
lactic  acid — viz.,  substances  which,  as  we  have  just  seen,  are  also 
found  in  muscle-tissue.  In  addition,  we  find  certain  albumins 
which  in  part  belong  to  the  native  albumins  and  in  part  to  the  globu- 
lins; further,  nucleins  and  albuminoids,  among  which  the  so-called 
neurokeratin  is  of  special  interest,  as  it  largely  enters  into  the  com- 
position of  the  supporting  structure  of  the  nerve-tissue,  and  is  char- 
acteristic of  this.  Besides  these  various  substances  nerve-tissue 
contains  especially  large  amounts  of  so-called  myelins — viz.,  lecithin, 
cholesterin,  and  protagon.  A  certain  amount  of  mineral  salts  and 
a  large  quantity  of  water  constitute  the  remaining  known  components 
of  the  tissue. 

As  regards  the  distribution  of  these  substances  among  the  gangli- 
onic cells  and  nerve-fibres  more  especially,  our  knowledge  is  as  yet 
quite  incomplete;  but  it  appears  from  the  analyses  which  are  avail- 
able that  the  gray  substance  of  the  brain  in  the  dried  state  consists 
to  the  extent  of  one-half  at  least  of  albumins,  while  the  substances 
which  arc  soluble  in  ether  amount  to  only  about  one-quarter  of  the 
total  quantity.  Protagon  is  here  present  in  only  very  small  quantity. 
In  the  white  substance  of  the  brain,  on  the  other  hand,  it  is  found 
in  considerable  amount,  while  the  albumins  constitute  only  one- 
quarter  of  the  dry  material.  In  embryonic!  brains,  in  which  the 
medullary  sheaths  are  not  us  yet  developed,  much  smaller  amounts 
of  lecithins  are  found  than  in  the  adult  brain  ;  and  it  is  noteworthy, 
moreover,  that  protagon  and  neurokeratin  are  hen;  both  absent.  It 
may  thus  be  concluded  that  these  substances  are  essentially  com- 
ponent- of  the  medullary  nerve-fibres.     According  to  Hoppe-Seyler, 

indeed,  the    small    amount  of   |>rota<_roii  which    is    found   in   the  gray 

Substance    of    the    brain    i-    entirely    referable    to    this    source.      The 

21  36S 


370  THE  NERVE-TISSUE. 

presence  of  smaller  amounts  of  lecithins  in  the  embryonic  brain, 
as  compared  with  the  adult  brain,  and  notably  the  gray  matter,  is 
now  generally  explained  upon  the  assumption  that  the  material 
in  question  is  in  some  manner  intimately  concerned  in  the  growth 
of  the  cellular  elements  proper. 

Analysis  of  Brain-tissue  (Baumstark). 

White  matter.1    Gray  matter.1 

Water 69.53  77.00 

Solids 30.47  23.00 

Insoluble  albumins  and  connective-tissue    .    .       5.00  6.08 

Neurokeratin 1.89  1.04 

Nucleins 0.29  0.20 

Protagon 2^51  L08 

Cholesterins,  free 1.82  0.63 

Cholesterins,  combined 2.69  1.75 

Mineral  salts 0.52  0.56 

Other  substances,  soluble  in  ether 30.47  23.00 

Analysis  of  the  Mineral  Salts  (Geoghegan). 

Chlorine 0.42  —  1.06 

Phosphoric  acid  (P04) 0.85  —  1.39 

Carbonic  acid  (C03) 0.25  —  0.33 

Sulphuric  acid  (S04) 0.14  —  0.13 

Phosphate  of  iron  (Fe2(P04)2) 0.09  —  0.30 

Calcium 0.02 

Magnesium 0.06  —  0.07 

Potassium 0.58  —  1.52 

Sodium 0.45  —  0.78 

Albumins. — Of  the  character  of  the  individual  albumins  which 
occur  in  nerve-tissue  very  little  is  known.  Baumstark  states  that 
they  are  essentially  the  same  as  those  of  muscle-tissue,  and,  to  judge 
from  the  researches  of  Petrowski,  it  appears  that  one  of  these  may 
be  identical  with  myosin,  as  it  is  soluble  in  dilute  saline  solution, 
and  can  be  precipitated  by  diluting  Avith  water  or  by  salting  with 
sodium  chloride.  The  coagulation-point  of  the  substance  has,  how- 
ever, not  been  determined.  It  is  said  to  occur  both  in  the  gray  and 
the  white  matter. 

More  recently  Halliburton  claims  to  have  isolated  two  neuro- 
globulins,  both  'from  the  white  and  the  gray  matter,  with  a  coagu- 
lation-point of  47°  and  75°  C,  respectively.  In  addition  he  found 
a  nucleo-albumin  in  the  gray  substance,  with  0.5  per  cent,  of  phos- 
phorus, which  coagulated  between  55°  and  60°  C.  v.  Jaksch 
further  claims  to  have  isolated  a  nuclein  from  the  gray  matter  which 
contains  but  little  phosphorus,  and  yields  hypoxanthin,  xanthin, 
phosphoric  acid,  and  an  albuminous  body  on  decomposition. ^ 

The  albumins,  as  I  have  indicated,  are  principally  found  in  the 
gray  matter  of  the  brain,  and  constitute  about  one-half  of  the 
dried  substance.  In  the  white  matter,  however,  they  are  also  found, 
though  in  mnch  smaller  amount,  and  it  is  thought  that  the  cylinder 

1  The  complete  separation  into  gray  and  white  matter  is,  of  course,  impossible. 


THE  NERVE- TISSUE.  371 

of  the  nerve-fibre  is  essentially  of  an  albuminous  nature.  They  are 
without  doubt  intimately  concerned  in  the  specific  function  of  the 
nerve-tissue,  but  of  the  part  which  they  take  in  such  function  noth- 
ing whatever  is  known.  It  is  interesting  to  note,  however,  that 
the  gray  matter  of  the  brain,  as  also  of  the  spinal  cord  and  groups 
of  ganglionic  cells  outside  of  the  central  nervous  system,  always  pre- 
sent an  acid  reaction,  while  the  white  matter  of  the  brain  and  cord 
and  the  peripheral  nerves  is  always  neutral  or  slightly  alkaline. 
The  substance  which  produces  the  acid  reaction  of  the  gray  matter 
is  apparently  the  common,  optically  inactive  lactic  acid,  and  it  is 
noteworthy  that  in  the  nerve-tissue  also  a  lactic  acid  is  encountered 
in  those  portions  which  are  especially  rich  in  albumins.  But  while 
the  acid  reaction  of  muscle-tissue  becomes  manifest  only  after  death, 
it  can  be  readily  shown  that  in  the  case  of  the  brain  and  spinal  cord 
this  is  normal  even  during  life.  Whether  or  not  other  substances 
besides  lactic  acid  contribute  to  the  acid  reaction  of  the  gray  matter 
has  not  been  definitely  established.  But  it  is  quite  likely  that 
this  is  the  case,  as  in  the  presence  of  lactic  acid  a  transformation  of 
diphosphates  to  monophosphates  would  of  necessity  occur.  Bibra 
and  Midler,  moreover,  claim  to  have  obtained  traces  of  formic  acid 
from  the  aqueous  extract  of  the  gray  matter.  Paralactic  acid  has 
not   been   found   in   nerve-tissue. 

Neurokeratin. — This  substance,  which  was  first  isolated  by 
Ki'ihne,  forms  the  greater  portion  of  the  supporting  tissue  of  the 
c  sural  nervous  system,  and  is  likewise  found  in  the  medullary 
fibres,  where  it  constitutes  the  axilemma  and  outer  sheath  of  the 
medullary  substance.  According  to  some  observers,  moreover,  it 
tonus  a  tine  reticulated  network  in  the  latter. 

Neurokeratin  is  an  albuminoid  and  belongs  to  the  group  of  the 
keratins,  which  are  found  widely  distributed  among  the  tissues  of 
epiblastic  origin.  In  the  invertebrate  animals,  in  which  medullary 
fibres  are  not  found,  and  chitinous  substances  largely  enter  into 
tli''  composition  of  the  outer  skeleton  of  the  body,  it  is  accordingly 
represented  by  a  neurochitin. 

Neurokeratin  is  insoluble  in  water,  ether,  alcohol,  in  dilute  solu- 
tion-of  tin'  alkaline  hydrates,  in  gastric  juice  and  pancreatic  juice. 
To  isolate  th"  substance  from  nerve-tissue,  this  is  accordingly  ex- 
tracted with  alcohol  and  ether,  to  remove  the  myelin  substances. 
The  remaining  material  is  freed  from  albumins  and  other  albuminoids 
by  digestion  with  i_ra-<tric  juice,  and  is  then  treated  with  a  dilute  solu- 

ti< t'  sodium   hydrate,  which  dissolves  the  nucleins.     The  keratin 

then  remains.  From  the  other  keratins,  which  may  be  obtained 
from  hair,  nail-,  horn-,  etc.,  neurokeratin  differs  especially  in  its  rela- 
tively -mall  amount  of   sulphur,  and  the  large  amount  of  carbon  and 

hydrogen   and   the  smaller  quantity  of  nitrogen  which  it   contains. 

This    18    shown  in    the    following    table,  which  is    taken    from    Ham- 

marsten  : 


372  THE  NERVE-TISSUE. 

Carbon.  Hydrogen.  Nitrogen.    Oxygen.  Sulphur. 

Human  hair   ....  50.66               6.36  17.14  20.85         5.00 

Nails         51.00              6.94  17.51  21.85         2.80 

Horn         50.86              6.94  .    .  .    .           3.30 

Egg-shell 49.78               6.56  16.43  22.90        4.25 

Turtle  shell      ....  54.89              6.94  16.77  19.56        2.22 

Neurokeratin  ....  56.11-58.45  7.26-9.02  11.50-14.32    .    .  1.63-2.24 

Its  *  properties  are  the  same  as  those  of  the  keratins  in  general 
(which  see). 

THE   MYELIN   BODIES. 

In  former  years  it  was  supposed  that  the  medullary  substance 
of  nerve-fibres  consisted  of  a  single  substance,  myelin,  which  was 
characterized  by  the  fact  that  on  treating  with  water  it  formed 
double-contoured  droplets,  which  can  readily  be  seen  on  microscopical 
examination.  According  to  Gad  and  Heymans,  this  myelin  is  in 
reality  lecithin  in  the  free  state  or  in  loose  combination,  and  we  know 
as  a  matter  of  fact  that  the  peculiar  reaction  is  due  to  decomposition- 
products  of  such  complex  substances  as  protagon  and  certain  com- 
pound cholesterins.  In  speaking  of  myelin  substances  at  the  present 
time  we  have  reference  to  protagon  s,  lecithins,  and  cholesterins. 

Protagon. — While  there  is  evidence  to  show  that  different  pro- 
tagons  exist,  wTe  are  not  as  yet  in  a  position  to  characterize  such 
forms  individually,  and  for  convenience'  sake  we  shall  speak  of 
protagons  as  a  chemical  unity  at  this  place.  The  substance  is  not 
strictly  characteristic  of  nerve-tissue,  as  it  has  also  been  found  in 
other  organs  of  the  body,  such  as  the  spleen,  in  the  stroma  of  the 
red  corpuscles,  in  pus,  and  in  spermatozoa.  But  while  it  is  here 
present  in  only  small  amounts,  it  enters  into  the  composition  of 
nerve-tissue  to  a  considerable  extent,  and  is  thus  quantitatively  at 
least  peculiar  to  these  structures.  Whether  or  not  the  substance 
occurs  also  in  the  gray  matter  appears  doubtful,  but  in  the  white 
matter  and  in  the  peripheral  medullated  nerve-fibres  it  is  abundant. 

According  to  Liebreich,  who  was  the  first  to  isolate  protagon 
from  brain-tissue,  the  substance  has  the  composition  C116H24)N4P02. 
It  is  to  be  noted,  however,  that  the  elementary  analysis  of  different 
preparations  has  given  rise  to  different  results,  which  in  itself  sug- 
gests the  probability  that  different  forms  exist.  According  to  some 
observers,  it  also  contains  sulphur  in  molecular  combination,  but 
recent  investigations  have  shown  that  this  is  probably  not  the  case. 

On  decomposition  with  boiling  baryta-water  protagon  yields  the 
same  products  as  the  lecithins,  viz.,  fatty  acids,  glycerin-phosphoric 
acid,  and  cholin.  But,  in  addition,  one  or  more  glucosides  are 
obtained,  which  have  been  termed  cerebrosides  by  Thudichum,  and 
of  which  three  are  now  recognized.  These  are  known  as  cerebrin, 
kerasin,  or  homocerebrin,  and  encephalin.  Others  also  may  possibly 
exist,  and  it  is  likely  that  the  pyosin  and  pyogenin,  which  Kossel 
and  Freitag  obtained  from  pus,  belong  to  this  order. 

On  boiling  with  dilute  mineral  acids  protagon  also  yields  a  re- 


THE  MYELIN  BODIES.  373 

ducing  substance,  which  is  commonly  regarded  as  galactose,  and  is 
referable  t»>  the  decomposition  of  the  glucosides  just  mentioned. 

Protagon  is  easily  soluble  in  warm  alcohol  and  ether,  while  in 
cold  alcohol  and  cold  ether  it  dissolves  with  difficulty.  On  cooling 
the  substance  crystallizes  out  in  tine  needles  or  in  waxy  masses, 
which  can  readily  be  broken  up  into  a  fine  powder.  On  heating  its 
alcoholic  solutions  to  a  temperature  of  48°  C.  or  on  boiling  its 
ethereal  solutions  the  substance  is  readily  decomposed  into  its  com- 
ponents, a-  indicated  above.  In  the  dry  state  it  can  be  heated  to 
a  higher  temperature,  but  it  is  then  also  decomposed  before  100°  C. 
is  reached.  The  resulting  products  melt  between  200°  and  203°  C, 
and  begin  to  volatilize  at  220°.  When  moistened  with  water,  the 
substance  swells  and  is  partly  decomposed,  with  the  formation  of 
so-called  "  myelin  "  droplets.  If  much  water  is  added,  an  opaque 
fluid  is  obtained. 

Isolation. — To  isolate  protagon  from  brain-tissue  this  should  be  as 
fresh  as  possible,  as  otherwise  partial  decomposition  occurs  spon- 
taneously. The  material  is  freed  from  its  membranes  and  adhering 
blood,  and  is  then  stirred  to  a  pulp,  and  extracted  with  85  per  cent. 
alcohol,  at  a  temperature  of  45°  C..  using  fresh  portions  of  alcohol 
from  time  to  time  until  a  specimen  no  longer  deposits  a  sediment 
when  cooled  to  0°  C.  The  extracts  are  filtered  at  45°  C,  and  sub- 
sequently kept  at  0°  C.  The  resulting  precipitates  are  extracted 
with  cold  ether  to  remove  cholesterins  and  lecithins,  when  the 
remaining  material  is  pressed  between  filter  paper  and  dried  over 
sulphuric  acid.  It  is  finally  pulverized, again  extracted  with  alcohol 
at  45°  C,  when  the  solution  is  filtered  and  cooled  to  0°  C.  To 
purify  the  substance  it  is  recrystaUized  from  warm  alcohol  or  ether. 

Cerebrin. — Cerebrin,  as  I  have  stated,  is  a  normal  decomposition- 
product  of  protagon,  but  probably  does  not  occur  in  the  living 
nerve-tissue  as  such.  Associated  with  lecithin,  it  is  also  found  in 
the  stroma  of  the  red  corpuscles  of  the  blood,  in  leucocytes,  in  sper- 
matozoa, in  the  spleen,  in  the  yolk  of  birds'  eggs,  etc.  It  is 
questionable,  however,  whether  it  actually  exists  in  the  free 
State,  and  the  fact  of  its  constant  association  with  lecithin  rather 
suggests  that    here   also  it   is   primarily   present   in    the    protagon 

molecule. 

Cerebrin  is  said  to  have  the  formula  ('7„III)IIX;,()I...  Elementary 
analysis  ha-  given  the  following  results:  C,  <i!).0<S  per  cent.; 
If,  11.17:  \.  2.13;  O,  L7.32.  On  decomposition  with  boiling 
mineral  acids  it  yields  a  reducing  substance  which  is  commonly 
regarded  a-  galactose.  On  oxidation  with  nitric  acid  or  on  fusion 
with  caustic  alkali  palmitic  acid  or  stearic  acid  is  obtained.  If 
tli<-  substance  is  dissolved  in  concentrated  sulphuric  acid,  the 
solution  gradually  assumes  a  purplish-red  color,  which  changes 
to  violet,  and  finally  to  brown.  On  adding  an  equal  amount  of 
water  and  boiling,  a  flocculent   precipitate  appears,  which   is  known 

a-     cdylid,    and     i-     -aid     to     have     the     composil ion    ( \  {\  I, .„,()_._    or 


374  THE  NERVE-TISSUE. 

(C16H3102)3.[C16H18(OH)3].  This  substance  supposedly  represents 
about  85  per  cent,  of  the  entire  weight  of  the  cerebrin.  It 
is  soluble  in  water,  in  hot  alcohol,  and  especially  in  ether  and 
chloroform.  On  fusion  with  caustic  alkali,  cetylid  yields  methane, 
hydrogen,  and  palmitic  acid. 

Pure  cerebrin  is  a  colorless  substance  which  occurs  in  the  form  of 
a  crystalline  powder,  consisting  of  microscopical  globulites.  It  is 
soluble  in  warm  acetone,  benzene,  chloroform,  and  boiling  alcohol, 
but  is  insoluble  in  ether,  even  at  its  boiling-point.  In  cold  water  it 
is  likewise  insoluble.  In  boiling  water  it  swells  to  a  certain  extent, 
like  starch  paste.  It  melts  at  177°  C,  but  is  decomposed  long 
before  with  the  development  of  a  yellow  or  brownish  color.  Its 
reaction  is  neutral. 

Isolation. — Cerebrin  can  be  obtained  either  from  protagon  after 
the  separation  of  the  latter  or  directly  from  the  brain  by  decompos- 
ing its  antecedents  with  baryta-water.  To  this  end,  the  material 
after  being  freed  from  its  membranes  and  blood  is  stirred  with 
baryta-water,  and  brought  to  the  boil.  The  insoluble  portion  is 
then  pressed  out  and  repeatedly  extracted  with  alcohol  by  boiling. 
The  extract  is  filtered  while  still  hot,  when  on  cooling  to  0°  C, 
the  cerebrin  separates  out  in  impure  form.  It  is  freed  from  choles- 
terin  and  fats  by  skaking  with  ether,  and  purified  by  repeated  solu- 
tion in  hot  alcohol  and  subsequent  cooling,  until  jelly-like  pre- 
cipitates, which  are  referable  to  homocerebrin  or  encephalin,  are  no 
longer  obtained. 

With  this  method,  however,  a  considerable  portion  of  the  cerebrin 
is  decomposed,  and  Kossel  has  accordingly  suggested  that  the 
protagon  be  first  extracted  and  subsequently  decomposed.  To  this 
end,  the  substance  is  dissolved  in  methyl  alcohol,  and  is  treated  with 
a  hot  methyl  alcoholic  solution  of  barium  hydrate.  The  mixture  is 
warmed  for  a  few  minutes  on  a  water-bath,  and  then  allowed  to 
cool.  The  resulting  precipitate  contains  the  entire  amount  of 
cerebrin  which  can  be  obtained,  and  represents  50  per  cent,  of  the 
original  quantity  of  protagon.  It  is  filtered  off,  suspended  in  water, 
and  freed  from  barium  by  a  current  of  carbon  dioxide.  The  insolu- 
ble residue  contains  the  cerebrosides,  which  are  now  extracted  with 
hot  alcohol  and  isolated  by  fractional  crystallization. 

Homocerebrin  (Kerasin). — The  formula  of  homocerebrin  is 
given  as  C70H138N2O12,  and  the  substance  could  hence  be  regarded  as 
an  anhydride  of  cerebrin.  In  the  dry  state  it  occurs  as  a  wax-like 
mass,  which  can  be  pulverized  only  with  difficulty.  From  its  solu- 
tion in  alcohol  and  boiling  ether  it  separates  out  in  aggregates  of 
exceedingly  fine  needles.  At  130°  C.  it  is  decomposed  with  the 
appearance  of  a  yellow  color,  and  melts  at  150°  C  Toward  con- 
centrated sulphuric  acid  and  water  it  behaves  like  cerebrin.  Like 
this,  it  yields  a  reducing  substance  of  the  character  of  galactose 
when  boiled  with  dilute  mineral  acids,  and  gives  rise  to  the  forma- 
tion of  cetylid,  like  cerebrin. 


THE  MYELIN  BODIES.  375 

The  amount  of  homocerebrin  which  may  be  obtained  from  pro- 
tagon  is  about  one-fourth  that  of  cerebrin.  It  is  isolated  in  associa- 
tion with  the  latter,  as  described,  and  can  be  separated  from  the 
cerebrin  by  fractional  crystallization  from  the  alcoholic  solution,  in 
which  it  is  more  readily  soluble  than  cerebrin. 

Encephalin. — Encephalin  is  regarded  as  a  transformation-product 
of  cerebrin,  and,  like  this,  yields  galactose  on  boiling  with  dilute 
mineral  acids.  Cetylid  is  also  obtained  on  treating  with  concentrated 
sulphuric  acid.  It  is  soluble  in  hot  alcohol,  but  tends  to  separate 
out  on  cooling  as  a  jelly-like  material.  On  slow  evaporation  it 
crystallizes  in  platelets,  which  melt  at  150°  C,  but  are  already 
decomposed  at  125°  C.  In  hot  water  it  swells  to  form  a  thick 
paste,  which  remains  on  cooling.  Like  homocerebrin,  the  substance 
is  found  in  the  alcoholic  solution  after  the  cerebrin  has  separated 
out  (see  above),  and  can  be  isolated  by  fractional  crystallization 
from  the  mother-liquor,  or  from  a  solution  of  acetone,  in  which  the 
homocerebrin  is  likewise  soluble. 

Lecithins. — The  lecithins,  which  may  be  isolated  from  nerve- 
tissue,  where  they  largely  exist  in  combination  with  the  cerebrosides, 
in  the  form  of  protagon,  but  undoubtedly  also  occur  as  such  in  the 
free  state,  are  as  yet  but  little  known.  On  decomposition  they 
yield  palmitic  acid,  stearic  acid,  oleic  acid,  glycerin-phosphoric  acid, 
and  cholin.  Of  their  significance  nothing  definite  is  known,  but  it 
appears  that  they  are  in  some  manner  intimately  concerned  in  the 
development  of  the  cells,  and  it  is  for  this  reason  probably  that 
much  smaller  amounts  can  be  obtained  from  the  embryonic  brain 
than  from  that  of  the  adult. 

(For  the  general  description  of  the  lecithins,  see  p.  65). 

Isolation. — To  isolate  the  lecithins  of  the  brain,  the  following 
method,  which  has  been  suggested  by  Ziilzer,  is  conveniently  em- 
ployed. The  brain,  which  must  be  perfectly  fresh,  is  freed  from 
membranes,  cut  into  thin  pieces,  and  placed  in  a  jar  with  ether. 
The  material  should  rest  on  a  layer  of  cotton.  After  standing  for 
several  days  the  ethereal  extract  is  poured  off,  and  separated  from 
the  lower  layer  of  blood.  The  extraction  is  continued  with  new  por- 
tions of  ether  so  long  as  anything  passes  into  solution.  If  desired, 
the  remaining  material  can  then  be  extracted  with  80  per  cent,  alco- 
hol ;it    15°  C,  which   takes    up   the   protagon,  as  has  been  described. 

The  ethereal  extracts  are  united  and  concentrated  in  the  vacuum. 
Any  protagon  that  lias  passed  into  solution  is  thus  thrown  down 
and  altered  off.  The  clear  solution  is  now  treated  with  an  excess 
of  acetone    SO    long   as    a    precipitate  is  formed.      This  is  filtered  off 

and  thoroughly  washed  with  acetone.  The  acetone-ethereal  solution 
we  term  A,  and  the  precipitate  15.     A,  contains  the  entire  quantity 

of  cholesterin.       To    recover   this,  the   acetone-ether    is    distilled    off, 

the  residue  ie  boiled  with  alcohol,  the  alcoholic  solution  is  filtered 
while  still  hot,  when,  on  cooling,  the  substance  crystallizes  out.  Its 
melting-point  Is  1  1-7   ( '. 


376  THE  NERVE-TISSUE. 

The  precipitate  B  is  placed  in  ether.  This  dissolves  the  greater 
portion,  while  a  smaller  amount  remains  undissolved.  The  latter 
consists  of  protagon,  which  was  previously  held  in  solution,  owing 
to  the  presence  of  cholesterin.  The  soluble  portion  is  treated  with 
alcohol  so  long  as  a  precipitate  forms.  This  precipitate  we  term  C, 
and  the  alcoholic  nitrate  D.  If  a  specimen  of  D  is  treated  with  an 
alcoholic  solution  of  platinum  chloride,  a  precipitate  results  ;  this 
is  not  abundant,  however,  and  consists  of  a  chloroplatinate  of  leci- 
thin. To  isolate  the  lecithins  as  such,  the  alcoholic  solution  is  pre- 
cipitated with  acetone,  or  the  ether-alcohol  is  distilled  off,  when  the 
lecithins  remain  as  a  tough,  wax-like  mass. 

Of  the  nature  of  the  substance  or  substances  which  are  contained 
in  the  precipitate  C,  nothing  definite  is  known.  Zulzer  apparently 
was  able  to  isolate  one  of  these,  however,  and  found  it  to  contain 
both  nitrogen  and  phosphorus.  He  suggests  that  it  may  possibly 
belong  to  the  so-called  cephalins  of  Thudichum.  But  of  the  nature 
of  these  also  our  knowledge  is  as  yet  insufficient  to  warrant  their 
description  at  this  place. 

The  Cholesterins. — Cholesterins  are  found  in  nerve-tissue,  both 
in  the  free  state,  and  as  so-called  combined  cholesterins,  but  of  the 
chemical    character  of  the   latter  we   are  as  yet  in  ignorance  (see 

P'67>. 

The  isolation  of  free  cholesterin  has  been  described  above. 

The  Extractives. — The  extractives  of  nerve-tissue,  as  I  have 
already  stated,  are  essentially  the  same  as  those  which  can  be 
isolated  from  other  organs  of  the  body.  They  comprise  traces  of 
kreatin,  uric  acid,  xanthin,  hypoxanthin,  guanin,  adenin,  inosit, 
volatile  fatty  acids  (acetic  acid  and  formic  acid),  lactic  acid,  glycogen, 
leucin  (or  rather  a  lower  member  of  the  homologous  series 
C2„H2n  +  jNO.,),  and  urea.  In  addition,  jecorin,  cholin,  and  neu- 
ridin  have  also  been  found ;  neurin,  on  the  other  hand,  does  not 
occur  in   the  brain  under  normal  conditions. 

Neuridin  is  of  special  interest,  as  the  substance  is  constantly 
formed  during  the  putrefaction  of  meat  and  gelatin,  and  has  also 
been  obtained  from  cultures  of  the  typhoid  organism.  According 
to  Brieger,  who  first  isolated  the  body,  it  is  also  present  in  traces  in 
the  yolk  of  birds'  eggs.  It  is  a  diamin  of  the  composition  C5H14N2. 
With  the  chlorides  of  gold  and  platinum  it  forms  well-defined 
crystalline  salts.  On  boiling  with  caustic  alkalies  it  is  decomposed 
into  trimethyl-amin  and  dimethyl-amin.     It  is  not  toxic. 

Jecorin  is  a  substance  of  unknown  composition,  but  apparently 
contains  both  sulphur  and  phosphorus.  It  is  not  exclusively  encoun- 
tered in  nerve-tissue,  but  has  also  been  found  in  traces  in  the  liver,  in 
the  muscles,  and,  according  to  some  observers,  in  the  blood.  It 
reduces  cupric  oxide  in  alkaline  solution  on  boiling,  and  on  cool- 
ing separates  out  in  the  form  of  a  thick  jelly.  It  is  soluble  in  ether, 
and  can  be  precipitated  from  its  solutions  by  alcohol. 


CHAPTER    XVIII. 

THE  EYE  AND  THE  EAR. 

THE  EYE. 

In  studying  the  chemical  composition  of  the  eye,  we  shall  con- 
sider the  most  important  parts  of  the  organ  in  succession.  It 
should  be  pointed  out  in  advance,  however,  that  the  subject  has 
thus  far  received  but  little  attention,  and  our  account  must  hence  of 
necessity  be  very  imperfect. 

The  Cornea. — An  analysis  of  the  cornea  of  the  ox  has  given 
the  following  results: 

Pro  mille. 

Water 758.3 

Solids 241.7 

Collagen 203.8 

Other  organic  substances      28.4 

Mineral  salts 9.2 

The  collagen,  which  forms  the  greater  portion  of  the  fibrous  net- 
work of  the  cornea,  is  probably  identical  with  the  common  form 
which  can  be  isolated  from  cartilage,  and,  according  to  Morner,  con- 
tains 16.95  per  cent,  of  nitrogen. 

The  semi  liquid  interfibrillary  substance  consists  of  a  mucoid, 
which  yields  a  reducing  substance  on  boiling  with  dilute  mineral 
acids.  It  contains  about  2  per  cent,  of  sulphur,  and  seems  to  be 
characteristic  of  corneal  tissue.  In  addition,  we  find  two  globulins, 
which,  according  to  Morner,  do  not  belong  to  the  cornea  proper, 
however,  but  are  contained  in  the  epithelial  layer.  Nucleins  have 
not  been  found. 

I)r.sf'f>inrt,.s  membrane  principally  consists  of  a  membranin,  which 
contains  14.77  per  cent,  of  nitrogen,  and  0.90  per  cent,  of  sulphur. 
The  substance  is  a  glucoproteid,  and  belongs  to  the  group  of  hyalo- 
gens  (which  see).  On  boiling  with  dilute  hydrochloric  acid  it  yields 
a  reducing  substance.  In  ordinary  boiling  water  it  is  insoluble,  but 
dissolves  under  the  action  of  superheated  steam.  It  is  digested 
by  trypsin,  while  the  gastric  juice  is  without  effect. 

The  Sclerotic. — The  composition  of  the  sclerotic  coat  of  the 
ey«-  i-  very  much  the  same  as  thai  of  the  cornea,  but  it  appears  that 
the  quantity  of  the  mucoid  is  here  much  less,  while  collagen  repre- 
sents about  seven-eighths  of  the  entire  amount  of  solids. 

The  Aqueous  Humor. — The  aqueous  humor  is  a  clear  fluid  of 
an  alkaline  reaction  and  a  specific  gravity  varying  between  1.003  and 

877 


378  THE  EYE  AND   THE  EAR. 

1.009.  Its  quantitative  composition  has  already  been  given  (page 
343).  According  to  Grunhagen,  it  contains  traces  of  paralactic  acid, 
a  dextrorotatory  body,  and  a  reducing  substance,  which  is  not  sugar. 
Both  the  latter  are  unknown.  The  albumins  in  question  are  serum- 
albumin,  serum-globulin,  and  traces  of  fibrinogen. 

The  Crystalline  Lens. — The  capsule  of  the  crystalline  lens, 
like  Descemet's  membrane,  consists  essentially  of  a  membranin, 
which  is  not  identical  with  that  found  in  the  latter,  however,  as  it 
is  less  resistant  to  the  action  of  boiling  water  and  of  acids  and  alka- 
lies. According  to  Morner,  it  contains  14.10  per  cent,  of  nitrogen 
and  0.83  per  cent,  of  sulphur. 

A  general  idea  of  the  chemical  composition  of  the  lens  itself  may 
be  formed  from  the  following  analysis,  which  I  have  taken  from 
Neumeister : 

Per  cent. 

Water 63.50 

Solids 36.50 

Albumins 35.00 

Insoluble  albuminoid 17.00 

/3-crystalline 11.00 

a-crystalline 6.80 

Albumin 0.20 

Fats 0.29 

Lecitbins      0.23 

Cholesterin 0.22 

Salts 0.80 

The  albumins  of  the  lens  can  be  divided  into  two  groups,  viz.,, 
those  which  are  soluble  in  dilute  saline  solution,  and  those  which  are 
insoluble.  The  latter  group  is  represented  by  a  substance  which  is 
spoken  of  as  albumoid.  It  is  manifestly  a  true  albumin,  as  it  is 
entirely  dissolved  by  the  gastric  juice,  and  does  not  yield  a  reducing 
substance  on  boiling  with  mineral  acids.  It  gives  all  the  common 
color  reactions  of  the  true  albumins,  and  has  the  same  elementary 
composition.  In  dilute  mineral  acids  and  alkalies  it  dissolves  with 
ease,  and  is  reprecipitated  on  neutralization.  Unlike  the  alkaline 
albuminates,  however,  its  solution  in  dilute  alkalies  coagulates  at 
50°  C,  in  the  presence  of  8  per  cent,  of  sodium  chloride.  The  sub- 
stance manifestly  constitutes  the  greater  portion  of  the  lens-fibres, 
as  the  nitrogen  and  sulphur  values  of  the  two  are  practically  the 
same,  viz.,  N,  16.62  and  S,  0.79  per  cent,  in  the  case  of  the  albumoid, 
as  compared  with  N,  16.61  and  S,  0.77  of  the  fibres.  It  can  be  shown, 
moreover,  that  after  extraction  of  the  soluble  constituents  of  the 
lens  the  fibrous  framework  remains  and  gives  the  same  reactions 
as  the  isolated  albumoid.  Its  amount  increases  from  without 
inward,  in  accordance  with  the  increasing  age  of  the  fibres. 

Aside  from  a  very  small  amount  of  serum-albumin,  the  remaining 
soluble  albumins  of  the  lens  are  represented  by  two  vitellins,  which 
are  termed  a-crystalline  and  /9-crystalline,  respectively.  Of  these, 
the  a-body  is  notably  found  in  the  outer  portion  of  the  lens, 
while  the  /5-substance  occurs  in  the  inner  portion  more  particularly, 


THE  EYE.  379 

and  is  apparently  the  only  one  that  is  found  in  the  centre  of  the 
lens. 

The  two  substances  can  be  isolated  from  an  aqueous  extract  of  the 
lens  by  saturating  the  solution  with  magnesium  sulphate  at  a 
temperature  of  30°  C.  The  precipitate  is  then  dissolved  in  water, 
dialvzed,  and  the  resulting  solution  precipitated  with  acetic  acid, 
which  throws  down  the  a-body,  while  the  /3-crystalline  remains  in 
solution.  A  small  amount  of  the  /9-substance,  it  is  true,  is  also  pre- 
cipitated by  the  acetic  acid,  but  can  be  separated  from  the  a-body  by 
a  repetition  of  the  process. 

Both  substances  are  precipitated  from  their  neutral  solutions  by 
carbon  dioxide,  but  in  the  case  of  the  ^-crystalline  this  precipitation 
is  never  complete.  The  latter  coagulates  at  63°  C,  and  the  a-crys- 
talline  at  72°  C.  The  ^-substance  further  differs  from  the  a-body 
in  containing  much  more  sulphur,  1.27  per  cent.,  as  compared  with 
0.56  per  cent.,  which  is,  moreover,  in  part  at  least,  present  in  a 
loosely  combined  form,  while  the  entire  quantity  that  is  found  in  the 
a-crystalline  is  firmly  combined. 

That  these  bodies  are  intimately  concerned  in  the  concentration  of 
the  light  cannot  be  doubted.  The  refractive  index  of  the  inner 
layer  of  the  lens,  in  man,  is  given  as  1.407,  while  that  of  the 
central  portion  is   1.456. 

Of  the  significance  of  the  fats,  lecithins  and  cholesterins  in  the 
lens,  nothing  is  known,  but  it  appears  that  the  amount  of  the  two 
latter,  at  least,  is  much  increased  in  senile  cataract,  while  the 
quantity  of  the  albumins,  as  a  whole,  is  diminished.  The  albumoid, 
however,  is  then  possibly  increased. 

The  Vitreous  Body. — The  vitreous  body  of  the  eye  is  a  jelly- 
like material,  which  consists  of  a  fine  framework  of  collagen,  enclos- 
ing the  liquid  portion  of  the  body  proper.  This  presents  an  alka- 
line reaction,  and  contains  only  a  very  small  amount  of  solids.  Its 
general  composition  is  seen  below  : 

Tro  mille. 

Water 989.00 

Solids 11.00 

Albumin 0.70 

Urea  .       0.04 

Paralactic  acid     traces. 

GrIucoBe      traces. 

Mineral  salts 9.00 

Among  tlic  albumins  present  Morner  claims  to  have  found  a 
hyalomucoid,  which  is  closely  related  to  the  cornea]  mucoid,  but 
contains  12.^7  per  cent  of  nitrogen  and  1.19  per  cent,  of  sulphur, 
a-  compared  with  12.79  per  cent.  <>('  nitrogen  and  2.07  percent,  of 
sulphur  in  the  case  of  the  latter. 

The  Retina. — A  general  idea  of  the  chemical  composition  of  the 
retina  may  lie  formed  from  the  following  analyses,  which  are  taken 
from  ( 'aim  : 


380  THE  EYE  AND  THE  EAR. 

Horse.  Ox. 

Water 89.99  86.52-87.61 

Solids 10.01  13.43-]  2.39 

Soluble  albumins 4.35  \  q  ak     7  no 

Insoluble  albumins 1.36/ 

Extractives 0.67  0.67-  1.07 

Cholesterin  ) 0.65-0.77 

Lecithin       [       2.39  2.08-  2.89 

Fats              )       0.00-  0.47 

Soluble  salts 1.11  0.67-  0.93 

Insoluble  salts 0.01  0.02-  0.27 

Like  the  gray  matter  of  the  brain,  from  which  the  retina  is  essen- 
tially derived,  the  membrane  presents  an  acid  reaction  when  per- 
fectly fresh,  but  becomes  alkaline  soon  after  death. 

The  albumins  which  are  found  in  the  retina  appear  to  be  identical 
with  those  of  the  brain  substance,  and  here,  as  there,  we  also  meet 
with  neurokeratin.  This  apparently  forms  the  sheath  of  the  outer 
portion  of  the  rods.  In  their  interior  we  meet  with  protagon, 
lecith-albumins,  and  in  many  animals  with  a  peculiar  red  pigment, 
which  has  been  termed  rhodopsin. 

Rhodopsin. — Of  the  significance  of  this  pigment  nothing  is  known. 
It  occurs  in  the  outer  portion  of  the  rods  and  is  absent  in  the 
cones.  As  the  macula  lutea,  viz.,  the  point  of  clearest  vision,  is 
composed  only  of  cones,  we  may  conclude  that  its  presence  is  not 
essential  to  sight,  and,  as  I  have  just  said,  the  pigment  is  not  found 
in  all  animals.  It  is  absent  in  chickens,  pigeons,  in  certain  reptiles, 
bats,  etc.,  but  is  present  in  owls  and  deep-sea  fishes.  On  exposure 
to  daylight  the  pigment  fades,  and,  to  isolate  the  substance,  it  is 
necessary  to  work  with  sodium  light.  If  the  living  retina,  after 
having  been  kept  in  the  dark  for  some  time,  is  suddenly  exposed  to 
an  intense  light,  which  is  broken  in  part  through  the  interposition 
of  some  dark  object,  such  as  the  framework  of  a  window,  and  if  the 
remaining  pigment  is  then  fixed  with  a  4  per  cent,  solution  of  alum, 
red  pictures  of  the  interposed  object  can  be  obtained  on  the  retina, 
while  the  remaining  portion  has  become  decolorized.  Such  pictures 
are  termed  optograms. 

The  regeneration  of  the  pigment,  which  is  constantly  going  on  in 
the  living  animal,  is  apparently  dependent  upon  the  integral  union 
of  the  layer  of  rods  and  cones  with  the  pigmented  epithelial  layer 
of  the  retina ;  but  of  the  manner  in  which  this  restitution  takes 
place,  we  know  nothing.  It  appears,  however,  that  its  formation  is 
preceded  by  the  development  of  a  yellow  pigment,  which  is  termed 
xanthopsin. 

Of  the  chemical  nature  of  rhodopsin  nothing  is  known.  Be- 
sides daylight,  it  is  decomposed  by  acids,  alcohol,  ether,  chloroform, 
and  solutions  of  the  alkaline  hydrates,  by  heating  to  a  temperature 
of  from  52°  to  53°  C.  for  several  hours,  or  instantaneously  at  76°  C. 
Toward  ammonia  and  a  solution  of  alum  it  is  refractory. 

It  is  easily  soluble  in  water,  containing  from  2  to  5  per  cent,  of 
Platner's  bile.     From  such  a  solution  it  is  precipitated  on  dialysis 


THE  EYE.  381 

or  by  salting  with  ammonium  sulphate  or  magnesium  sulphate.  The 
substance  then  appears  as  a  violet  amorphous  material.  On  spec- 
troscopic examination  no  specific  bands  are  observed,  but  merely  a 
general  absorption  between  D  and  C,  which  is  especially  marked 
about  E. 

Chromophanes. — Chromophanes  are  pigments  which  apparently 
belong  to  the  lipochromes,  and  are  found  in  the  retinal  cones  of 
reptiles  and  bin  Is.  They  here  occur  in  the  form  of  red,  green,  and 
yellow  oil  globules,  which  are  quite  distinct,  and  are  situated  at  the 
inner  ends  of  the  cones.  The  pigments  in  question  are  termed  rhodo- 
phane,  chlorophane,  and  xanthophane,  respectively.  Unlike  the 
pigment  of  the  rods,  these  chromophanes  are  apparently  not  affected 
by  light,  unless  exposed  for  several  days.  Of  their  significance, 
nothing  is  known. 

The  epithelial  layer  of  the  retina,  which  adjoins  the  choroid,  con- 
tains a  black  pigment,  which  is  probably  identical  with  that  of  the 
choroid.  This  is  termed  fusdn,  and  belongs  to  the  class  of  the  mel- 
anins,  which  comprise  the  black  pigments  that  are  found  in  the  hair, 
in  the  negro-skin,  in  melanotic  tumors,  etc.  According  to  Lan- 
dolt,  this  particular  form  contains  C,  54.48  per  cent.;  H,  5.36 ; 
X.  12.65;  O,  27.52.  In  addition  iron  is  also  found,  and  the 
opinion  has  been  expressed  that  the  substance  may  be  derived  from 
the  coloring-matter  of  the  blood.  This  supposition  is  strengthened 
by  the  observation  that  the  first  appearance  of  the  pigment  in  the 
embryo  coincides  in  point  of  time  writh  the  development  of  the 
choroidal  bloodvessels. 

Besides  fuscin,  a  pigment  of  a  yellow  color  has  also  been  found 
in  the  pigmented  epithelial  lining  of  the  retina,  which  is  termed 
lipochrin.     It  is  apparently  a  yellow  lipochrome. 

The  Choroid. — Aside  from  the  common  components  of  connec- 
tive tissue,  the  choroid,  as  has  just  been  mentioned,  contains  a  black 
pigment,  fuscin,  which  is  probably  identical  with  that  found  in  the 
pigmented  epithelial  lining  of  the  retina  (see  above). 

THE   EAR. 

The  chemical  composition  of  the  organic  portion  of  the  middle 
arid  the  internal  ear  has  not  as  yet  been  studied.  The  perilymph 
and  endolymph  present  an  alkaline  reaction,  and,  in  addition  to  the 
common  mineral  -alt-  of  the' lymph,  contain  traces  of  albumin,  and 
in  aome  animals  a  mucinous  body  of  unknown  character. 


CHAPTER*  XIX 


THE  SUPPOKTING  TISSUES. 


In  contradistinction  to  those  tissues  of  the  animal  body  which  are 
essentially  composed  of  cells,  and  in  which  the  albumins  proper  con- 
stitute the  greater  portion  of  the  organic  solids,  we  find  that  in  the 
so-called  supporting  tissues  which  comprise  the  common  connective 
tissues,  cartilage,  and  bone,  the  albuminoids  stand  in  the  foreground. 
Their  preponderance  here  coincides  with  the  extensive  development 
of  the  matrix,  while  the  cellular  elements  enter  into  the  histological 
picture  to  a  more  or  less  insignificant  extent.  This  statement,  how- 
ever, holds  good  only  for  the  higher  animals,  and  more  specifically 
for  the  fully  developed  animals.  In  lower  forms  of  life,  and  during 
the  embryonic  stage  of  the  development  of  the  higher  forms,  these 
structures  are  rich  in  cells,  and  we  find  then  an  underlying  matrix 
in  which  a  differentiation  into  supporting  tissue  proper  has  not  as 
yet  occurred  or  exists  to  only  a  limited  extent.  Such  tissue  is 
termed  embryonic  connective  tissue,  and  is  also  known  as  mucous 
tissue.  In  the  adult  animal  it  is  found  only  in  the  vitreous  humor 
of  the  eye.  In  typical  form  it  is  seen  in  the  umbilical  cord,  in 
which  it  constitutes  the  so-called  jelly  of  Wharton.  The  matrix  is 
here  very  rich  in  water,  and  contains  a  mucinous  substance,  which 
is  soluble  in  a  0.5  pro  mille  solution  of  hydrochloric  acid.  In  addi- 
tion, traces  of  albumin  are  met  with,  while  collagen  is  usually  absent. 
Of  the  composition  of  the  cells  nothing  specific  is  known,  but  it  is 
quite  likely  that  their  processes  consist  of  collagen. 

White  Fibrous  Tissue. — The  fibrils  of  white  fibrous  tissue  con- 
sist of  collagen,  and  are  bound  together  by  a  cement-substance, 
which  represents  the  original  undifferentiated  matrix.  As  in  the 
case  of  the  embryonic  connective  tissue,  this  contains  traces  of  the 
common  albumins  of  the  plasma,  and  a  mucinous  substance,  which, 
in  contradistinction  to  that  of  the  umbilical  cord,  is  insoluble  in 
a  0.5  pro  mille  solution  of  hydrochloric  acid.  Analysis  of  this 
substance  has  given  the  following  results:  C,  48.30;  H,  6.44;  N, 
11.75  ;  S,  0.81  ;  and  O,  32.70  per  cent.  According  to  Lobisch,  its 
formula  is  C160H256N32SO80.  To  isolate  the  body  in  question,  liga- 
ments, such  as  the  tendo  Achillis,  are  cut  into  small  pieces  and 
first  extracted  with  cold  water,  which  dissolves  the  albumins  and 
a  small  fraction  of  the  mucin.  The  remaining  material  is  then 
placed  in  a  half-saturated  solution  of  lime-water,  in  which  the 
mucin  is  readily  soluble.  After  filtering,  the  substance  is  precipitated 
by  adding  an  excess  of  acetic  acid  (see  page  113).    The  residual  sub- 

382 


CARTILAGE.  383 

stance,  after  removal  of  the  mucin,  consists  of  the  collagen-fibrils, 
a  few  cellular  elements,  and  quite  commonly  also  contains  isolated 
fibrils  of  the  yellow  or  elastic  variety,  which  can  be  readily  recog- 
nized on  microscopical  examination  by  their  higher  power  of  refrac- 
tion. When  plaeed  in  water,  or,  still  better,  in  a  dilute  solution  of 
acetic  acid  or  caustic  alkali,  the  white  fibres  swell,  while  solutions 
of  some  of  the  metallic  salts,  such  as  ferric  sulphate  and  mercuric 
chloride,  cause  them  to  shrink.  Tannic  acid  acts  in  a  similar 
manner.  Owing  to  the  great  stability  of  the  compound  of  the  latter 
with  collagen,  tannic  acid  is  extensively  utilized  in  the  preparation 
of  leather. 

On  boiling  white  fibrous  tissue  in  water  the  collagen  dissolves, 
with  thf  formation  of  gelatin,  which  latter  separates  as  a  jelly-like 
mass  on  cooling. 

Yellow  or  Elastic  Tissue. — In  the  yellow  elastic  fibres,  elastin 
takes  the  place  of  the  collagen  of  the  white  fibrous  variety.  For 
purposes  of  study,  the  substance  is  most  conveniently  obtained  from 
the  ligamentum  nuchas  of  the  ox,  in  which  such  fibres  are  almost 
exclusively  found  (see  page  47). 

Reticulated  Tissue. — In  the  reticulated  tissue,  which  constitutes 
the  fibrous  framework  of  the  lymph-glands  of  the  body,  but  which 
is  also  found  in  the  alveoli  of  the  lungs,  in  the  liver,  the  kidneys, 
and  the  intestinal  mucous  membrane,  the  fibres  consist  of  reticulin. 

Reticulin  is  said  to  have  the  composition  C,  52.88  ;  H,  6.97  ;  N, 
15.63;  S,  1.88;  P,  0.34.  It  is  insoluble  in  water,  alcohol,  ether, 
dilute  mineral  acids,  lime-water,  and  solutions  of  sodium  carbonate. 
It  resists  the  action  of  pepsin  and  trypsin,  and  is  dissolved  only  in 
cold  .-odium  hydrate  solution  on  standing  for  several  weeks.  It 
docs  not  give  Millon's  reaction,  and  accordingly  yields  no  ty  rosin  on 
hydrolytic  decomposition.  On  prolonged  boiling  with  water  or 
dilute  alkalies,  its  phosphorus  is  split  off;  the  residual  material  is 
then  Boluble  in  water,  and  can  be  precipitated  from  its  solutions  by 
means  of  acetic  acid. 

CARTILAGE. 

Histologically  considered,  cartilage  consists  of  a  more  or  less 
hyalin  matrix,  in  which  a  variable  nimiber  of  cartilage-cells  are  found 
imbedded  In  certain  localities,  further,  a  differentiation  of  the  mat- 
rix into  fibres,  both  of  the  white  and  the  yellow  clastic  variety,  is 
observed.  Such  fibres,  as  in  the  case  of  the  corresponding  connec- 
tive tissue,  COnsisf  of  collagen  and  elastin,  respectively. 

Of  the  composition  of  the  cells  nothing  specific  is  known.  Appar- 
ently they  contain  a  small  amount  of  glycogen,  which  disappears 
during  starvation.  Traces  of  fat  are  also  found.  During  embryonic 
life  they  are  quite  numerous,  but  later  they  diminish  in  number,  and 
in  the  adult  animal  the  matrix  largely  predominates.  Embryonic 
cartilage  doe-  not  yield  gelatin  on  boiling  with  water,  and  it  is  quite 

likely  that    a-    in    the   cage   of  the    matrix    of  embryonic   connective 


384  THE  SUPPORTING   TISSUES. 

tissue  the  matrix  here  also  consists  essentially  of  water  and  some 
mucinous  substance.  Whether  or  not  this  is  identical  with  the  so- 
called  chondromucoid,  which  can  be  obtained  from  the  cartilage  of 
the  adult  animal,  is  not  known. 

A  general  idea  of  the  chemical  composition  of  cartilage  may  be 
formed  from  the  following  analyses,  which  are  taken  from  His  : 

Costal  cartilage  Articular  cartilage 

(human).  from  knee-joint  (human). 

Water 67. 67  per  cent.  73.59  per  cent. 

Solids 32.33   "       "  26.41    "       " 

Organic  material    ....      30.13   "       "  24.87    "       " 

Mineral  salts 2.20   "       "  1.54   "       " 

Analysis  of  the  mineral  salts  has  given  the  following  results 
(calculated  for  100  parts  of  the  mineral  ash): 

Sodium  chloride 6.11    per  cent.  22.48  per  cent. 

Sodium  sulphate 44.81       "       "  55.17    "       " 

Potassium  sulphate 26.66      "       "  .    . 

Sodium  phosphate      8.42      "       "  7.39   "       " 

Calcium  phosphate 7.88  \  u       u  ..__..„       IC 

Magnesium  phosphate       .    .    .        4.55  j 

The  organic  constituents  of  the  cartilaginous  matrix  are  essentially 
represented  by  chondroitin-sulphuric  acid  as  such,  and  its  compounds 
with  collagen  and  albumins.  In  addition,  a  small  amount  of  soluble 
albumins  is  found,  as  also  a  peculiar  insoluble  albuminous  sub- 
stance, which  has  been  termed  albumoid. 

Chondroitin-sulphuric  Acid. — This  substance  is  a  conjugate 
sulphate,  and,  according  to  Schmiedeberg,  has  the  composition 
C18H2rN014.S03.  On  hydrolytic  decomposition  it  yields  a  hyalin, 
chondroitin,  which  in  turn  gives  rise  to  the  formation  of  chondrosin, 
and  this  to  glucuronic  acid  and  glucosamin,  as  represented  by  the 
equations : 

(1)  C18H27NOu.S03   +   H20  =   C18H27NOu   +   H2S04 

Chondroitin-sulphuric  Chondroitin. 

acid. 

(2)  C18H27NOM   +  3H20         =  C12H2,NOn    +  3CH3.COOH 
Chondroitin.  Chondrosin.  Acetic  acid. 

(3)  Cl2H21NOH  +   H20  =   COOH(CH.OH)4.COH    +  C6Hn05.NH2 
Chondrosin.  Glucuronic  Glucos- 

acid.  amin. 

Both  chondroitin  and  chondrosin  are  monobasic  acids.  The  latter 
reduces  Fehling's  solution  directly,  while  in  the  case  of  chondroitin 
this  occurs  only  after  the  substance  has  been  decomposed. 

Chondroitin-sulphuric  acid  is  an  amorphous  substance,  and  is 
soluble  in  water.  A  concentrated  solution  resembles  mucilage  in 
appearance  and  consistence.  Its  salts  are  also  for  the  most  part 
soluble  in  water.  The  sodium  and  potassium  salts  can  be  pre- 
cipitated by  means  of  ferric  chloride,  lead  subacetate,  and  alcohol, 
while  silver  nitrate,  zinc  chloride,  tannic  acid,  and  potassium  ferro- 
cyanide,  the  latter  in  the  presence  of  acetic  acid,  are  without  effect. 


CARTILAGE.  385 

In  the  cartilage  its  potassium  and  sodium  salts  occur  both  as  such 
and  in  combination  with  collagen  and  albumins.  A  mixture  of  these 
compounds,  according  to  Schmiedeberg,  constitutes  the  so-called 
chondromucoid  of  Morner.  If  a  solution  of  gelatin  is  mixed  with 
an  acidified  solution  of  the  potassium  or  sodium  salts  of  the  acid, 
a  precipitate  occurs.  This  also  results  if  cartilage  is  boiled  with 
water  and  the  resulting  impure  solution  of  gelatin,  which  was 
formerly  termed  chondrin,  is  acidified  with  a  dilute  mineral  acid. 
The  precipitate  consists  of  the  free  chondroitin-sulphuric  acid,  and 
is  soluble  in  an  excess  of  the  acid. 

Chondroitin-sulphuric  acid,  while  essentially  a  constituent  of  car- 
tilage, has  also  been  found  in  other  organs  of  the  body,  as  in  the 
inner  coats  of  the  larger  arteries,  in  the  kidneys,  and  under  patho- 
logical conditions  in  amyloid  livers.  Traces  are  likewise  found  in 
the  urine. 

Isolation. — To  isolate  chondroitin-sulphuric  acid  from  cartilage 
shavings,  the  material  is  boiled  with  a  5  per  cent,  solution  of  caustic 
alkali.  The  solution  is  neutralized,  and  freed  by  filtration  from 
the  alkaline  albuminates  which  have  been  formed  during  the  process 
of  boiling.  Albumoses  are  removed  by  means  of  tannic  acid,  the 
excess  of  the  latter  by  means  of  lead  subacetate,  and  the  excess 
of  lead  with  hydrogen  sulphide.  The  acid  is  then  precipitated  with 
alcohol.  To  purify  the  substance,  it  is  dissolved  in  water,  and  the 
solution  dialyzed  and  reprecipitated  with  alcohol.  The  solution  in 
water  and  precipitation  with  alcohol  is  repeated  several  times,  when 
the  acid  is  finally  washed  with  alcoholic  ether. 

Isolation  of  Chondromucoid. — To  isolate  the  chondromucoid, 
viz.,  the  compounds  of  chondroitin-sulphuric  acid  with  collagen 
and  albumins,  the  cartilage  shavings  are  first  extracted  with  water, 
which  dissolves  the  free  chondroitin-sulphuric  acid  and  a  small 
amount  of  the  chondromucoid.  On  acidulating  this  solution  with 
a  •')  pro  mille  solution  of  hydrochloric  acid  and  heating  on  a  water- 
bath,  the  chondromucoid  is  gradually  precipitated,  while  the  free 
acid  remains  in  solution.  The  cartilaginous  residue  is  then  ex- 
tracted  with  a  2  to  3  pro  mille  solution  of  hydrochloric  acid  at  a 
temperature  ol*  .'>o°  to  40°  C,  which  dissolves  any  collagen  that 
may  be  presenl  a-  such.  Alter  washing  with  water  the  remaining 
material  i-  extracted  with  a  5  pro  mille  solution  of  caustic  alkali. 
The  chondromucoid  i~  thus  dissolved,  and  is  then  precipitated  with 
an  acid.  After  repeated  solution  in  an  alkali  and  precipitation 
with  an  acid  it   i-  finally  washed  with  alcohol  and    ether. 

Albumoid. — The  albumoid  which  is  found  in  the  cartilage  of 
adult  animals  is  apparently  closely  related  to  elastin  and  keratin, but 
differs  from  the  latter  iii  containing  sulphur,  and  from  the  former  in 
its  digestibility  by  gastric  juice.  It  gives  the  common  color-reac- 
tions of  the  albumins,  bul  is  insoluble  in  all  neutral  solvents,  and 
dissolves  in  acid-  and  alkalies  only  with  great  difficulty. 

Isolation. — To    isolate  the  Bubstance,  cartilage  shavings  arc  first 


386  THE  SUPPORTING    TISSUES. 

extracted  with  a  0.5  per  cent,  solution  of  caustic  alkali,  to  remove 
the  chondromucoid,  and  the  free  chondroitin-sulphuric  acid.  The 
remaining  material  is  then  washed  with  water  and  boiled  with 
water  in  a  Papin  digester.  Any  collagen  that  may  be  present  is 
thus  dissolved,  while  the  albuminoid  together  with  the  cartilage-cells 
remains  behind. 

Mineral  Constituents.— Among  the  mineral  constituents  of  car- 
tilage, the  very  large  amount  of  alkaline  sulphates  is  especially 
noteworthy.  These  are  supposedly  not  present  in  the  free  state, 
however,  beyond  traces  perhaps,  but  result  from  the  chondroitin- 
sulphate  on  incineration.  In  the  cartilage  of  the  shark,  very  curi- 
ously, sodium  chloride  constitutes  as  much  as  94.2  per  cent,  of  the 
total  amount  of  mineral  ash.  As  this  represents  17.7  per  cent,  of 
the  moist  material,  the  amount  of  sodium  chloride  would  be  suffi- 
cient to  form  a  concentrated  solution  in  the  cartilage,  which,  of 
course,  is  scarcely  conceivable  as  occurring  in  living  tissue.  It  is 
hence  assumed  that  the  salt  is  present  in  organic  combination,  but 
of  its  pairling  nothing  definite  is  known.  According  to  Bunge,  such 
large  amounts  of  sodium  chloride  are  also  found  in  mammals  during 
the  period  of  intra-uterine  life  and  shortly  after  birth,  and  he  be- 
lieves that  this  is  in  accordance  with  the  biogenetic  law  Mrhich  under- 
lies the  development  of  the  higher  forms  of  life  from  those  of  a 
lower  order. 

With  the  appearance  of  old  age  a  gradual  deposition  of  calcium 
salts  occurs  in  the  matrix  of  the  cartilage,  so  that  partial  ossifica- 
tion takes  place.  This,  of  course,  also  occurs  during  the  develop- 
ment of  normal  bone,  but  it  is  to  be  noted  that,  in  contradistinction 
to  true  bone,  the  matrix  of  senile,  ossified  cartilage  retains  its 
original  characteristics. 

BONE. 

The  matrix  of  bone-tissue,  like  its  contained  fibrils,  is  com- 
posed of  collagen,  which  is  here  termed  ossein,  and  is  supposedly 
identical  with  the  common  form  that  is  obtained  from  connective 
tissue.  Of  the  composition  of  the  cells,  viz.,  the  so-called  bone- 
corpuscles,  nothing  is  known.  With  their  processes  they  occupy  the 
lacunae  and  canaliculi,  and  are  separated  from  the  bony  structure 
proper  by  a  layer  of  a  very  resistant  albuminous  substance  of 
unknown  character. 

In  contradistinction  to  the  other  supporting  tissues  of  the  body 
which  have  thus  far  been  considered,  bone-tissue  apparently  con- 
tains no  glucoproteids. 

The  function  of  the  bone-tissue,  as  the  principal  supporting  tissue 
of  the  body,  finds  its  expression  in  the  preponderance  of  the  mineral 
constituents  over  the  organic  solids,  and  it  is  interesting  to  note  that 
the  ratio  between  the  two  is  fairly  constant,  not  only  in  different 
bones,  but  also  in  different  animals.  These  salts  are  largely  repre- 
sented by  calcium  phosphate  and  carbonate,  which   impregnate  the 


BONE. 


387 


entire  matrix.  In  addition,  we  find  magnesium  phosphate  and 
small  amounts  of  calcium  chloride,  calcium  fluoride,  potassium  and 
sodium  salts,  and  a  little  iron.  Of  the  manner  in  which  the  differ- 
ent -alt-  are  combined  with  each  other,  nothing  definite  is  known, 
but  we  may  possibly  assume,  with  Gabriel,  that  the  composition  of 
the  bone-ash,  as  well  as  the  tooth-ash,  can  be  represented  by  the 
formula  [Ca,(P04)2  +  Ca5HP3013  +  H20],  in  which  2  to  3  per  cent. 
of  calcium  is  replaced  by  magnesium,  potassium,  and  sodium,  and 
4  to  b'  per  cent,  of  the  phosphoric  acid  by  carbonic  acid,  chlorine, 
and  fluorine. 

Whether  or  not  the  mineral  constituents  of  the  bone  exist  in  com- 
bination with  the  organic  components  of  the  tissue  has  not  as  yet 
been  definitely  ascertained,  but  does  not  appear  improbable. 

An  idea  of  the  quantitative  distribution  of  the  different  salts _  in 
different  animals  and  bones  may  be  formed  from  the  accompanying 
analyses.  The  figures  have  reference  to  100  parts  of  bone-ash 
(Zalefsky) : 


Calcium  phosphate  (Ca3(P04).2)      .    . 

Magnesium  phosphate  (Mg3(P04)2)    . 

Calcium   in  combination  with  carbon 

dioxide,  chlorine,  and  fluorine     .    . 

Carbon  dioxide1 

Chlorine 

Fluorine2 


Small  flounder  (ash  in  general)  . 

Man  fash  in  general) 

.Man  (  humerus) 

<  )x  I  lemur)      

ash  in  general) 

Babbits,  varying  in  age  between 
one  day  and  four  years  (general 
asb  | 


Human. 

Ox.            Turtle 

Guinea- 
pig- 

83.89 

86.09        85.98 

87.38 

1.04 

1.02          1.36 

1.05 

7.65 

7.36           6.32 

7.03 

5.73 

6.20           5.27 

1.80 

2.00           .    . 

2.30 

3.00          2.00 

Calcium 

Phosphoric 

Magnesium 

oxide 

acid 

oxide 

(CaO). 

(PA). 

(MgO). 

53.13 

42.72 

0.91 

52.83 

38.73 

0.48 

51.31 

36.65 

0.77 

51.28 

37.46 

1.05 

51.01 

38.19 

1.27 

51.91-52.89 

39.78-42.20 

0.83-1.38 

The  variations  in  the  amount  of  bone-ash,  as  a  whole,  in  different 
bones  of  the  same  animal,  are  seen  in  the  following  table  (Fremy): 

Per  cent. 

Femur 

Humerus     

Tibia f  64.1-64.6 

Occipital  bone I 

Cranium J 

Scapula 63.3 

Vertebrae 54.2 

The  ; 1 1 1 1 ' » 1 1 1 i  1  of  water  which  is  found  in  bones  varies  between  13.8 
and  1  L3  per  cent.  It  is  greater  in  the  spongy  bones  than  in  those 
of  the  compact  variety,  and  gradually  diminishes  with  age. 

1  These  figures  are  lomewhal  too  low,  us  a  certain  amount  of  carbon  dioxide  escapes 
during  the  incineration  of  the  bone. 

-  according  to  Uabrlel,  the  amount  of  fluorine  does  not  exceed  u.i  percent.,  and  is  usually 
less  than  0.5  per  cent. 


388  THE  SUPPORTING   TISSUES. 

The  bone-marrow  is  pervaded  by  a  network  of  connective  tissue, 
which  is  partly  of  the  white  fibrous  variety  and  partly  reticulated. 
In  its  meshes  we  find  the  cellular  elements  of  the  marrow,  viz., 
the  so-called  myeloplaxes,  the  juvenile  forms  of  the  polynuclear 
neutrophilic  and  eosinophilic  leucocytes,  viz.,  the  myelocytes,  red  cor- 
puscles in  various  stages  of  development,  and  a  variable  number  of 
fat  cells.  These  latter  are  especially  numerous  in  the  so-called  yellow 
marrow,  where  the  amount  of  fat  may  represent  as  much  as  96  per 
cent,  of  the  entire  substance.  It  consists  of  olein,  palmitin,  and 
stearin.  The  red  marroiv,  on  the  other  hand,  contains  a  much  smaller 
amount  of  fat,  and  owes  its  color  to  large  numbers  of  red  corpuscles. 
It  contains  albumins,  of  which  one  is  regarded  as  a  globulin,  and  is 
said  to  coagulate  at  50°  C.  But  especially  interesting  is  the  pres- 
ence of  peculiar  iron  compounds  which  are  as  yet  but  little  known, 
but  probably  belong  to  the  nucleo-albumins  and  iron-containing 
albuminates.  Their  presence  is  no  doubt  intimately  associated  with 
the  formation  of  red  corpuscles.  Among  the  extractives  of  bone- 
marrow  we  notably  find  lactic  acid  and  hypoxanthin. 

THE    TEETH. 

The  dentin  of  the  teeth  is  a  peculiarly  modified  form  of  bone- 
tissue,  and  is  likewise  composed  of  an  organic  matrix,  which  con- 
sists of  collagen  and  is  impregnated  with  mineral  salts.  The  latter 
are  here  even  more  abundant  than  in  true  bone,  and  represent 
from  64  to  68  per  cent,  of  the  fresh  tissue,  while  of  organic  matter 
we  find  between  26  and  28  per  cent.,  thus  leaving  10  per  cent, 
for  water.  The  relative  distribution  of  the  individual  salts  is  about 
the  same  as  in  common  bone. 

The  dentinal  tubules,  like  the  lacunas  and  canaliculi,  are  appar- 
ently lined  by  the  same  albuminous  substance. 

The  cement  which  surrounds  the  dentin  of  the  root  as  far  as 
the  neck  of  the  tooth   consists  of  true  bone. 

The  enamel,  in  accordance  with  its  epithelial  origin,  contains  no 
collagen.  It  is  very  rich  in  lime  salts,  and  its  mineral  constituents 
represent  as  much  as  96  per  cent,  of  the  total  substance.  Its  organic 
components  correspond  to  about  3.6  per  cent.,  but  are  as  yet 
unknown.     Water  is  practically  absent. 

Other  tissues  which  are  closely  related  to  bone  are  ivory,  tortoise- 
shell,  and  the  scales  of  fishes.  In  the  two  former  the  mineral  con- 
stituents predominate  over  the  organic  matter,  as  in  bone,  while  in 
the  latter  more  organic  material  is  found.  Ivory  is  especially  rich 
in  magnesium  phosphate,  of  which  it  contains  about  15.72  per  cent. 
The  outer  surface  of  tortoise-shell  is  covered  with  a  layer  of  kera- 
tinized epidermis. 

In  the  invertebrate  animals,  with  the  exception  of  the  cephalopods, 
and  possibly  also  the  branchiopods,   collagen  is  not    found.     The 


ADIPOSE  TISSUE.  389 

internal  supporting  structures  are  here  represented  by  ingrowths  of 
the  cutieular  formations,  which  are  derived  from  the  epidermal  cells, 
and  consist  largely  of  skdetins  and  hyabgens  which  have  become 
impregnated  with  lime  salts.  Closely  related  to  the  latter  is  chitin, 
which  enters  largely  into  the  composition  of  the  outer  _  skeleton 
of  the  arthropods  and  their  nerve-sheaths.  Associated  with  it  we 
find  the  so-called  bmiein,  which  is  regarded  as  a  cellulose,  and  which 
is  found  in  especial  abundance  in  the  mantle  of  the  tunicates. 

ADIPOSE    TISSUE. 

Adipose  tissue  may  be  regarded  as  a  special  form  of  connective 
tissue  in  which  the  cellular  elements  enclose  globules  of  fat.  AVhen 
fully  developed,  the  individual  cells  appear  as  greatly  distended 
vesicles,  which  are  covered  by  a  cell-membrane.  The  original  proto- 
plasm has  been  almost  entirely  replaced  by  fat,  and  occurs  merely 
as  a  thin  laver  beneath  the  membrane.  The  nucleus  also  has 
been  displaced  to  the  periphery,  and  can  scarcely  be  discerned  with- 
out special  methods  of  staining.  Such  fat-cells  usually  occur  in 
-roups,  and  are  held  together  by  delicate  fibres  of  connective  tissue, 
in  the  meshes  of  which  a  network  of  blood-capillaries  is  found 
surrounding  each  cell.  When  large  numbers  of  fat-cells  occur, 
the  individual  groups  are  gathered  into  lobules,  and  these  into 
lobes.  In  the  living  tissue  the  contained  fat  exists  in  a  liquid 
form,  but  congeals  after  death,  and  is  then  more  or  less  solid  accord- 
ing to  the  character  of  the  individual  fats.  Stearin  and  palmitin 
then  often  separate  out  in  crystalline  form.  Of  the  chemical  cora- 
position  of  the  original  cells/ before  their  invasion  with  fat-globules, 
oothing  definite  is  known.  They  contain  albumin  and  are  appar- 
ently rich  in  water.  The  cell-membrane  is  exceedingly  resistant  to 
solvents,  but  is  digested  by  the  gastric  juice,  and  possibly  consists 
of  an  elastin-like  substance. 

The  relative  amount  of  water  and  fat  which  is  found  in  adipose 
tissue  varies  primarily  with  the  state  of  nutrition,  and  differs  in 
ditl'Tent  animal-. 

Tie-  fats  in  question  are  principally  the  triglycerides  of  stearic 
acid,  palmitic  acid,  and  oleic  acid.  Others,  such  as  the  pjlycerides 
ofcapronic  acid  and  valerianic  acid,are  ool  constant  constituents  of 
adipose  tissue,  but  are  met   with   only  exceptionally,  and  always  in 

very  small   ai mts.      In    man,  a   comparatively   large  amount  oi 

olein  is  found,  but  it  is  not  BO  abundant  as  in  certain  cold-blooded 
animals,  in  which  it    may  form  the  greater  portion  of  the  fat.     The 

quantitative  relation    between    the    three    forms  is  by  00  means   con- 

-mni  in  all  parts  of  the  body,  so  that  the  melting-poinl  of  the  fats 
from  differenl  regions  may  be  quite  different.  It  differs,  moreover, 
in  different  animals.     This   ie  shown  in  the  following  table,  which 

is  taken  fr<»m  Gautier  : 


390  THE  SUPPORTING   TISSUES. 

Mutton  (subcutaneous)       27°-31°  C. 

Mutton  (perirenal)      37°-43°  C. 

Mutton  (epiploic)        36°-39°  C. 

Man  (panniculus  adiposus) 15°-22°  C. 

Man  (perirenal )      ' 25°  C. 

Dog 20°-22.5° 

Ox      39°  C. 

Bone-marrow  of  ox 45°  C. 

Calf 52°  -f  C. 

Horse 31°  +  C. 

Pig 40°  C. 

Duck      35°  C. 

Of  special  interest  is  the  fact  that  it  is  possible  to  replace  the  com- 
mon fats  of  one  animal  by  those  of  another,  and  even  by  fats  which 
are  not  found  normally  in  the  animal  world.  If  dogs,  in  which  the  fats 
have  been  removed  by  starvation,  are  thus  fed  with  vegetable  fats,  such 
as  rape-oil,  this  is  subsequently  found  in  the  tissues  of  the  animal, 
and  may  be  recognized  by  its  low  melting-point  (23°  C.)  and  the 
presence  of  the  glyceride  of  erucic  acid.  In  a  similar  manner  a 
deposition  of  mutton  tallow  may  be  effected,  which  begins  to  melt 
at  about  40°  C,  while  the  common  fat  of  dogs  melts  at  20°  C. 

In  addition  to  the  fats,  small  amounts  of  lecithin,  cholesterin,  and 
free  fatty  acids  may  also  be  isolated  from  adipose  tissue.  We 
further  find  a  yellow  lipochrome,  to  which  the  color  of  the  fat  is 
due. 

Analysis  of  Adipose  Tissue. — The  material  in  question  is  first 
dried,  ground  with  a  little  sand,  and  extracted  successively  with 
ether  and  alcohol.  The  alcoholic  extract  is  evaporated  to  dry- 
ness, the  residue  washed  with  water  to  remove  the  soluble  salts,  and 
is  then  extracted  with  ether.  The  ethereal  extracts  are  united,  and 
the  ether  is  distilled  off,  when  the  fats,  lecithins,  cholesterins,  free  fatty 
acids,  and  the  lipochromes  remain.  The  fatty  acids  are  transformed 
into  their  salts  by  adding  a  slight  excess  of  sodium  carbonate,  and 
heating  to  a  temperature  of  100°  C.  The  resulting  soaps  are  ex- 
tracted with  water.  The  insoluble  portion  is  dissolved  in  ether,  the 
ether  is  distilled  off,  and  the  residue  is  heated  on  a  water-bath  with 
an  alcoholic  solution  of  sodium  hydrate,  which  saponifies  the  fats. 
The  resulting  material  is  extracted  with  ether,  which  dissolves  the 
cholesterin.  The  insoluble  residue  is  dissolved  in  water,  and  the 
solution  is  saturated  with  carbon  dioxide  and  extracted  with  strong 
alcohol.  This  takes  up  the  soaps  and  the  glycerin.  The  alcoholic 
solution  is  transformed  into  an  aqueous  solution,  in  which  the  soaps 
are  decomposed  with  a  dilute  acid.  The  free  fatty  acids  are  thus 
precipitated,  and  can  then  be  separated  from  each  other  according 
to  the  usual  methods. 

Aside  from  adipose  tissue,  fats  are  met  with  in  all  the  organs  of 
the  body,  but,  with  the  exception  of  the  mammary  glands  during 
their  functional  activity,  they  are  found  normally  only  in  traces. 
Under  pathological  conditions,   however,  notable  quantities  of  fat 


ADIPOSE  TISSUE.  391 

may  be  met  with.  AVe  then  speak  of  a  fatty  degeneration  of  the 
organs.  This  is  especially  observed  in  the  liver  in  cases  of  acute 
yellow  atrophy,  and  can  also  be  brought  about  artificially  by  poison- 
ing with  phosphorus,  antimony,  arsenic,  etc. 

Among  the  fluids  of  the  body,  large  quantities  are  normally 
found  only  in  the  milk,  and  in  the  chyle  during  the  process  of 
digestion. 

Origin  of  the  Fats. — That  a  portion  of  the  fat  that  is  found 
in  the  animal  body  is  directly  referable  to  the  fats  which  have  been 
ingested  as  such  cannot  be  doubted.  This  is  proved  not  only  by 
the  observation  that  it  is  possible  to  replace  the  fats  which  are 
peculiar  to  a  certain  animal  by  those  of  another,  or  even  by  vegetable 
'ats,  as  has  been  shown  above,  but  also  by  the  fact  that  a  gradual 
deposition  of  fats  occurs  in  dogs  which  have  been  starved  and 
are  then  fed  on  very  little  albuminous  material,  but  with  much  fat. 
1.1  such  cases  it  can  easily  be  proved  that  the  amount  of  albu- 
mins invested  is  far  too  small  to  be  the  source  of  the  fat  that  has 
been  stored. 

The  ingested  fats,  however,  are  not  the  only  source  of  the  fats 
found  in  the  tissues,  and  there  is  evidence  to  show  that  they  may 
also  be  derived  from  the  albumins  and  the  carbohydrates.  Their 
origin  from  the  former  is  suggested  by  many  observations.  It 
is  thus  well  known  that  the  albuminous  constituents  of  human 
bodies  when  buried  in  moist  ground,  and  notably  the  muscle-tissue, 
may  undergo  a  peculiar  transformation,  which  is  characterized  by 
the  disappearance  of  the  albumins  and  their  replacement  by  free 
fatty  acids  and  the  calcium  and  ammonium  soaps  of  palmitic  acid 
and  -tearic  acid,  which  constitute  the  so-called  adipoaere,  or  Leichen, 
wax  of  the  Germans.  This  transformation,  however,  is  probably 
brought  about  through  the  activity  of  micro-organisms,  and  does  not 
prove  in  itself  that  in  the  living  animal  an  actual  formation  of 
neutral  fats  can  occur  from  the  albumins.  But  it  shows,  at  all 
event-,  that  forces  which  are  at  work  in  the  living  world  can  bring 
about  the  formation  of  two  of  the  higher  fatty  acids  at  least  which 
enter  into  the  composition  of  the  common  fat  from  albuminous 
material.  In  the  laboratory  such  a  transformation  has  not  as  vet 
been  accomplished,  if  we  disregard  the  observations  of  E.  Voit,  who 
claims  to  have  noted  the  appearance  of  higher  fatty  acids,  when  43 
grammes  of  albumin  were  kept   in    milk  of  lime  for  twelve  months. 

Proof  of  the  possible  origin  of  fats  from  albumins,  on  the  other 
band,  seems  to  be  afforded  l,v  the  phenomena  of  fatty  degenera- 
tion, where  an  actual  deposition  of  large  amounts  of  fat  can  be 
demonstrated  in  the  cell-  of  organs  in  which  only  traces  are  nor- 
mally found.  It  has  been  urged,  however, thai  the  fat  which  is  here 
encountered  has  not  developed  /'//  situ,  but  has  been  carried  to  the 
organs  in  question  from  the  adipose  tissue  proper.  That  such  a 
transposition  of  Cat-  may  occur  is  indeed  possible,  bul  it  has  been 
conclusively  shown  that  large  quantities  of  fin  may  be  isolated  from 


392  THE  SUPPORTING    TISSUES. 

the  liver  of  dogs  which  have  been  poisoned  with  phosphorus  after 
having  previously  fasted  for  twelve  days.  In  such  an  event  it 
scarcely  seems  admissible  to  attempt  to  account  for  the  presence  of 
the  large  amounts  of  fat  in  the  liver  on  the  theory  of  a  transposi- 
tion. Bauer,  moreover,  has  pointed  out  that  while  in  such  cases  a 
largely  increased  elimination  of  nitrogen  occurs,  there  is  evidence  to 
show  that  a  non-nitrogenous  portion  of  the  albuminous  molecule  is 
retained,  as  the  absorption  of  oxygen  and  the  elimination  of  carbon 
dioxide  are  decreased  by  one-half. 

A  further  proof  of  the  possible  origin  of  fats  from  albumins  has 
been  furnished  by  Hofmann.  Experimenting  with  maggots  of 
flies,  he  determined  the  amount  of  fat  in  one  portion  directly,  and 
then  permitted  a  second  portion  of  the  same  weight  to  develop  in 
defibrinated  blood  containing  a  known  amount  of  fat.  These  were 
then  killed  and  analyzed.  The  result  showed  that  they  contained 
an  amount  of  fat  which  was  from  seven  to  eleven  times  as  large 
as  the  total  amount  of  fat  in  the  blood,  plus  the  amount  which  they 
originally  contained. 

Of  the  manner  in  which  the  fat  originates  from  albuminous 
material  we  know  nothing  definite,  but  we  may  assume  that  non- 
nitrogenous  groups  are  here  first  split  off,  and  that  the  fats  are  then 
formed  from  these  synthetically.  That  an  actual  liberation  of  fatty 
radicles  can  occur  directly  appears  very  unlikely,  as  we  have  no 
evidence  whatever  to  show  that  the  albuminous  molecule  contains 
any  radicles  with  more  than  six  or  nine  atoms  of  carbon.  Of  the 
nature  of  these  non-nitrogenous  groups  we  know  nothing.  We 
have  shown,  however,  that  most  albumins  contain  a  carbohydrate 
group,  and  that  glucose  and  glycogen  can  both  be  derived  from 
that  source.  The  question  hence  suggests  itself,  Is  it  possible  that 
the  formation  of  fats  from  albumins  takes  place  with  the  inter- 
mediary formation  of  carbohydrates?  As  a  matter  of  fact,  there 
is  evidence  to  show  that  this  may  occur,  as  the  possible  origin  of 
fats  from  carbohydrates  is  now  well  established.  This,  transforma- 
tion represents  one  of  the  most  important  synthetic  phenomena 
which  occur  in  the  animal  world,  and  is  of  the  nature  of  a  synthetic 
reduction,  in  which  the  CHOH  groups  of  the  carbohydrates  are 
transformed  into  CH2  groups. 

That  fats  may  actually  be  formed  from  carbohydrates  can  be 
demonstrated  in  various  ways.  Two  animals  from  the  same  litter 
and  approximately  of  equal  weight  are  starved  until  the  stored  fat 
has  mostly  disappeared.  The  one  is  then  killed  so  as  to  ascertain 
the  amount  of  albumins  and  fats  which  still  remains.  The  other 
animal  is  now  fed  with  a  definite  quantity  of  some  cereal,  the  con- 
tained amounts  of  albumins,  starch,  and  fat  of  which  are  known. 
The  feces  are  carefully  collected  and  the  amount  of  non-resorbed 
fat  and  albumins  ascertained.  After  a  variable  period  of  time  this 
animal  is  also  killed,  and  the  amount  of  albumins  and  fat  esti- 
mated.    The  increase  in  the  amount  of  albumins  must,  of  course, 


ADIPOSE  TISSUE.  393 

he  referable  to  that  ingested,  while  the  increase  in  the  fat  may  be 
in  part  due  to  the  ingested  fat,  in  part  to  albumins,  and  in  part  to 
carbohydrates.  In  such  cases  it  has  been  found  that  the  amount  of 
both  albumins  and  fat  which  were  contained  in  the  food  are  by 
no  means  sufficient  to  account  for  all  the  accumulated  fat,  so 
that  the  conclusion  is  unavoidable  that  a  certain  proportion  of  this 
must  be  referable  to  the  ingested  carbohydrates.  Calculation  has, 
indeed,  shown  that  as  much  as  86.7  per  cent,  of  the  fat  is  of  such 
origin. 

Significance  of  the  Fats. — As  regards  the  function  of  fat  in  the 
animal  body,  our  knowledge  is  still  very  imperfect.  Owing  to  its 
property  as  a  poor  conductor  of  heat,  it  is  probably  of  moment 
in  preventing  an  undue  irradiation.  Its  principal  significance,  how- 
ever, is  undoubtedly  connected  with  its  manifest  value  as  a  food-stuff 
and  as  a  source  of  energy.  But  we  do  not  know  whether  this  is 
expressed  in  any  specific  function  of  the  body  beyond  the  produc- 
tion of  heat  in  general.  As  the  fat  disappears  during  starvation 
before  the  albumins  are  attacked,  we  may  assume  that  its  presence 
under  normal  conditions  prevents  an  undue  destruction  of  those  ele- 
ments which  essentially  represent  the  living  tissue.  In  this  respect, 
however,  the  fats  are  inferior  to  the  carbohydrates,  as  is  apparent 
from  the  fact  that  in  starving  animals  the  administration  of  fats  does 
not  lead  to  so  marked  a  diminution  in  the  elimination  of  nitrogen 
as  is  effected  by  a  corresponding  amount  of  carbohydrates.  Upon 
this  basis  also  Voit  lias  explained  the  well-known  phenomenon  that 
herbivorous  animals  are  more  likely  to  accumulate  albumins  than  the 
carnivora,  as  the  latter  receive  scarcely  any  carbohydrates  in  their 
food,  while  that  of  the  former  contains  comparatively  large  amounts. 


CHAPTER     XX. 

THE  SKIN   AND   ITS   APPENDAGES. 

In  considering  the  chemical  composition  of  the  skin  and  its 
appendages,  we  shall  deal  more  exclusively  with  those  substances 
which  are  more  or  less  peculiar  to  its  epidermal  structures.  The 
remaining  components  have  already  been  studied  in  detail  and 
require  no  further  consideration  at  this  place. 

The  epidermal  structures  in  the  case  of  the  vertebrate  animals 
comprise  the  epithelial  lining  of  the  skin,  together  with  its  sweat 
glands,  the  sebaceous  glands  and  allied  glands,  the  hair,  the  nails, 
the  hoofs,  the  feathers,  etc. 

On  section  of  the  epidermis  we  discern  different  layers  of  cells. 
The  lowest  of  these,  which  is  known  as  the  Malpighian  layer,  is 
composed  of  the  youngest  cells,  from  which  all  others  are  derived. 
These  are  distinctly  protoplasmic  in  character,  but  with  increasing 
age  they  become  dry  and  scaly,  and  are  finally  represented  by  fine 
lamellas  of  keratin,  which  are  constantly  thrown  off  and  regenerated 
from  below.  Keratin,  itself,  however,  is  not  found  in  the  lower 
layers  of  the  epidermis,  as  has  been  shown  by  Ernst,  but  appears 
only  above  the  so-called  stratum  granulosum.  In  the  latter  we 
find  peculiar  granules,  which  are  scattered  about  the  nuclei  of  the 
cells,  and  which  Ernst  regards  as  derivatives  of  the  nuclei.  They 
are  known  as  eleidin  granules,  and,  according  to  some  observers, 
represent  intermediary  products  in  the  formation  of  keratin.  Of 
their  chemical  nature,  however,  nothing  is  known. 

The  transition  of  the  soluble  albumins  of  the  lower  strata  of  cells 
into  the  insoluble  keratin  also  finds  its  expression  in  the  greater 
resistance  which  the  upper  layers  offer  to  the  action  of  the  caustic 
alkalies.  For,  while  the  lower  cells  are  dissolved  with  comparative 
ease  the  upper  strata  are  scarcely  affected  by  the  reagent.  Pancreatic 
juice  and  gastric  juice  behave  in  the  same  manner,  and  it  is  thus 
possible  to  separate  the  insoluble  keratin  from  the  soluble  albumins 
which  may  be  present  at  the  same  time.  Like  the  horny  layer  of 
the  skin,  so  also  are  other  epidermal  structures,  such  as  nails, 
hair,  horn,  feathers,  etc.,  largely  composed  of  keratin.  In  addi- 
tion, we  find  a  variable  amount  of  salts,  among  which  the  insoluble 
forms  are  especially  important.  Their  presence  manifestly  serves 
the  purpose  of  increasing  the  rigidity  of  these  structures.  Espe- 
cially noteworthy  is  the  large  amount  of  silicic  acid  which  is  found 
in  the  hair  and  in  feathers.     Besides  this,  we  meet  with  variable 

394 


THE  SKIN  AXD  ITS  APPENDAGES.  395 

amounts  of  phosphates  and  sulphates  of  the  alkalies  and  alkaline 
earths,  and  very  curiously  also  with  iron  salts.  The  fact  that  larger 
amounts  of  the  latter  are  found  in  dark-colored  hair  than  in  hair 
of  a  lighter  color  suggests  that  their  presence  may  be  dependent 
upon  these  pigments. 

The  black  and  brown  pigments  which  are  found  in  the  hair  and 
in  the  skin  of  the  negro  belong  to  the  group  of  the  mdanins. 
Individually  these  bodies  are  but  little  known,  and  it  is  an 
open  question  whether  the  iron  that  is  found  in  the  ash  is  present 
in  these  structures  in  molecular  combination  with  the  pigments. 
Unlike  the  fuscin  of  the  choroid  and  the  hippomelanin  that  has  been 
obtained  from  melanotic  tumors  in  horses,  the  melanins  of  the  skin 
and  the  hair  arc  easily  soluble  in  solutions  of  the  alkaline  hydrates. 
They  contain  sulphur  (2  to  4  per  cent.),  but  not  in  so  large  amounts 
as  the  phymatorhusin  that  has  been  isolated  from  melanotic  growths 
of  man  and  from  the  urine  (8  to  10  per  cent.). 

Into  the  various  pigments  which  have  been  found  in  the  skin  of 
reptiles,  in  the  scales  of  fishes,  in  the  feathers  of  birds,  etc.,  it  is 
scarcely  necessary  to  enter  at  this  place.  They  partly  belong  to  the 
melanin-,  partly  to  the  lipochromes  ;  others  are  classified  as  mela- 
noids,  while  still  others  are  closely  related  to  the  haemoglobins.  To 
a  certain  extent,  an  >reover,  the  colors  of  birds'  feathers  appear  to  be  of 
a  physical  nature  and  referable  to  certain  phenomena  of  interference. 

In  the  invertebrate  animals  various  pigments  are  also  observed, 
but  are  for  the  most  part  unknown.  The  keratin,  as  I  have  already 
stated,  is  here  represented  by  other  tegumentary  substances,  such  as 
chitin,  tuniciu,  the  hyalogens,  and  the  skeletins  (which  see). 

The  Sweat. — The  sweat  is  the  specific,  secretory  product  of  the 
corresponding  glands,  which  are  found  imbedded  in  the  lower  portion 
of  the  dermis.  In  man,  these  glands  rank  next  in  order  to  the 
kidney.-  in  importance  as  excretory  organs  of  water,  and  arc  capa- 
ble, to  a  certain  extent  at  least,  of  assuming  a  vicarious  activity 
when  the  kidneys  are  diseased.  In  man  their  number  is  quite 
large,  exceeding  2,000,000.  Their  distribution,  however,  is  not 
uniform,  and  as  a  result  there  are  certain  portions  of  the  body 
in  which  a  more  abundant    secretion    is   noted  than  in  others.      Such 

ions  an-  the  forehead,  the  armpits,  the  palms  of  the  bands,  the 
Bolea  of  the  feet,  etc. 

Anion'/  the  mammalian  animal-,  however,  some  exist  in  which 
a  secretion  of  sweat  docs  not  occur.  This  is  the  case  in  many 
of  the  rodent-  and  the  goat.  The  sheep,  the  horse,  and  the  ape-. 
on  the  other  hand,  sweat  over  their  entire  body,  while  other  animals, 
like  the  eat  and  the  dog,  BWeal  only  from  the  balls  of  the  tor-. 

The  amount  ,,("  -went  excreted  in  man  is  very  variable.  It  differs 
in  different  individuals  ;  and  i-  dependent  upon  the  surrounding 
temperature,  the  amount  of  water  ingested,  the  temperature  of  the 
body,  the  amount  of  exercise  taken,  etc.  It  is  manifestly  under  the 
control  of*  the  central  -nervous  system,  and  is  increased  by  painful 


396  THE  SKIN  AND  ITS  APPENDAGES. 

sensations  and  emotions,  by  stimulation  of  the  sciatic  nerve,  follow- 
ing the  administration  of  pilocarpin,  etc. 

Ordinarily  the  secretion  of  sweat  is  scarcely  noticeable,  as  the 
droplets  evaporate  almost  as  rapidly  as  they  appear  on  the  sur- 
face of  the  skin.  But  even  so,  from  700  to  900  c.c.  of  water  are 
daily  eliminated  by  the  body.  Artificially  this  amount  can  be 
greatly  increased,  and  it  is  stated  that  from  6000  to  8000  c.c.  may 
be  excreted  in  the  twenty-four  hours  if  the  body  is  kept  at  a  tem- 
perature of  from  40°  to  50°  C,  and  large  amounts  of  fluid  are 
ingested. 

Sweat,  recently  secreted,  is  more  or  less  turbid,  owing  to  an  admix- 
ture of  desquamated  epithelial  cells,  and  droplets  of  fat,  which 
are  in  part  derived  from  the  sebaceous  glands,  but  are  to  some 
extent  also  referable  to  the  sweat-glands  proper.  After  filtration  it 
appears  as  a  clear,  transparent,  colorless  fluid,  of  a  salty,  somewhat 
acrid  taste,  and  a  very  characteristic  odor,  which  differs  somewhat 
according  to  the  region  of  the  body  from  which  the  sweat  is  derived. 
Its  specific  gravity  varies  between  1.004  and  1.005. 

At  the  beginning  of  its  secretion  the  sweat  presents  an  acid  reac- 
tion, which  is  probably  referable  to  an  admixture  of  fatty  acids 
derived  from  the  sebaceous  glands  ;  later,  however,  it  is  alkaline. 

Under  normal  conditions  the  sweat  is  essentially  a  very  dilute 
aqueous  solution  of  mineral  salts,  but  in  addition  we  also  find  small 
amounts  of  many  urinary  components,  such  as  urea,  uric  acid, 
kreatinin,  aromatic  oxy-acids,  volatile  fatty  acids,  skatoxyl  and 
phenol  sulphate,  besides  fat,  cholesterin,  traces  of  albumin,  salts  of 
lactic  acid,  and  so-called  sudoric  or  hydratic  acid,  etc.  Larger 
amounts  of  solids  are  principally  met  with  in  cases  of  renal  insuffi- 
ciency, and  it  may  then  happen  that  the  elimination  of  urea  through 
the  sweat  increases  to  10  grammes,  as  compared  with  0.043—1.55 
pro  mille,  which  may  be  regarded  as  normal.  In  some  cases  of 
this  kind  the  urea  may  actually  be  found  in  the  form  of  a  fine  cry- 
stalline powder  deposited  all  over  the  skin.  Glucose  has  been 
observed  in  diabetes.  Cystin  has  been  noted  in  cases  of  cystinuria, 
and  abnormally  large  amounts  of  uric  acid  have  been  found  in 
gout.  In  jaundice  bilirubin  may  color  the  sweat  a  bright  yellow. 
A  blue  and  red  color,  which  is  thought  to  be  referable  to  the 
presence  of  indigo-blue  and  indigo-red,  has  also  been  observed 
(chromhidrosis  or  cyanhidrosis)  ;  and  it  is  stated  that  in  rare 
instances  blood  may  appear  in  the  sweat  (hsemahidrosis).  It  is 
noteworthy,  furthermore,  that  a  number  of  foreign  substances, 
when  ingested  by  the  mouth,  such  as  quinin,  the  various  salts 
of  iodine,  mercury,  and  arsenic,  are  in  part  eliminated  in  the 
sweat. 

A  general  idea  of  the  quantitative  composition  of  the  sweat  may 
be  formed  from  the  accompanying  analyses,  which  are  taken  from 
Favre,  Schottin,  and  Funke. 


THE  SKIN  AND  ITS  APPENDAGES. 


397 


Sweat  in  gen- 
eral (obtained 
by  elevation 
of  tempera- 
ture). 
Favre. 

Water 995.573 

Solids  .  . 4.427 

Soluble  in  water : 

Sodium  chloride 'J. 2.30 

Potassium  chloride 0.244 

Alkaline  sulphates 0.012  \ 

Alkaline  phosphates traces  J 

Albuminates 0.005 

Insoluble  in  water,  but  soluble  in  acidu- 
lated water  : 

Earthy  phosphates traces 

Soluble  in  alcohol  : 

Alkaline  lactates 0.3171 

Alkaline  sudorates 1.562  ! 

Urea 0.043  j 

Fats  and  fatty  acids  .    ......  0.014  J 

Insoluble  in  water  and  alcohol : 

Epithelium traces 


Sweat 

(from   extremities). 


SchSttin. 

977.40 

22.60 

3.6    1 
1.31 


Funke. 

988.40 

11.60 


4.36 


0.39  J 


11.30 


4.20 


7.24, 
of  which 
1.55  urea. 

2.49 


Gases. — While  in  mammals  and  birds  the  respiratory  function  of 
the  skin  is  insignificant  as  compared  with  that  of  the  lungs,  we  find 
that  in  the  amphibia  life  may  persist  for  quite  a  while  after  removal 
of  the  lungs,  and  that  during  this  time  oxygen  is  actively  taken  up 
from  the  air  and  carbon  dioxide  eliminated  in  turn.  If,  however, 
the  exchange  of  gases  is  impeded  or  prevented  by  covering  the 
skin  with  a  thin  layer  of  varnish,  death  rapidly  takes  place.  This 
also  occurs,  it  is  true,  in  some  of  the  smaller  mammals  which  have 
a  delicate  skin,  but  it  is  now  known  that  the  fatal  end  is  here  not 
referable  to  the  impairment  of  the  cutaneous  respiration,  nor  to  a 
retention  of  waste  products,  as  was  formerly  supposed,  but  to  a 
paresis  of  the  cutaneous  vasomotor  nerves  and  a  resulting  dilatation 
of  the  bloodvessels.  As  a  result  an  abnormally  increased  irradia- 
tion of  heat  occurs,  which  constitutes  the  direct  cause  of  death.  If 
tin-  is  prevented  by  placing  the  animal  in  a  warm  chamber  or  by 
surrounding  it  with  cotton  and  the  like,  recovery  may  take  place, 
in  the  larger  mammals,  including  man,  in  which  a  coarser  skin 
exists,  no  deleterious  effects  are  noted  even  after  ten  days. 

The  amounl  of  carbon  dioxide  which  is  given  off  by  man  through 
the  skin  is  principally  dependent  upon  the  surrounding  temperature, 
and  varies  between  8. }  grammes  at  29°  to  33°  C,  and  28.8  grammes 
ai  38.5    C. 

The  Sebum. — The  sebum  is  the  specific  secretory  product  of  the 
Bebaceous  glands,  and  serves  the  purpose  of  a  lubricant.  Amounts 
sufficient  foY  analytical  purposes  can  be  obtained  from  newly  born 
children,  in  which  the  secretion  constitutes  the  so-called  vernix  case- 
osa.  In  the  fresh  state  it  is  a  semiliquid,  oily  material,  in  which 
on  microscopical  examination  can  be  discerned  desquamated  epithe- 
lial cells  in  various  stages  of  degeneration,  fal  droplets,  fatty  acid 
needle-,  and  quite  constantly  also  plates   of  cholesterin.     Almost 


398  THE  SKIN  AND  ITS  APPENDAGES. 

immediately  on  exposure  to  the  air  it  solidifies  to  a  white,  tallow-like 
material.     Its  reaction  is  alkaline. 

The  most  important  constituents  of  the  sebum,  as  also  of  the 
related  secretion  of  the  uropygian  gland  of  birds,  are  the  compound 
eholesterins  (see  page  67).  Their  presence  has  been  demonstrated 
on  the  feathers  and  bills  of  birds,  in  the  wool  of  sheep,  in  the  hair  of 
mammals,  on  the  spikes  of  porcupines,  etc. ;  and  as  these  bodies  are 
remarkably  resistant  to  the  influence  of  putrefactive  organisms,  it 
has  been  supposed  that  their  presence  on  the  skin  and  its  append- 
ages serves  the  purposes  of  protecting  the  exposed  portions  of  the 
body  against  bacterial  invasion. 

In  addition  to  substances  of  this  order,  the  sebum  contains  a  vari- 
able amount  of  fats,  fatty  acids,  soaps,  lecithins,  mineral  salts,  and 
at  least  two  albumins,  of  which  one  is  commonly  regarded  as  casein. 

Closely  related  to  the  common  sebum  of  the  sebaceous  glands  of 
the  skin  proper  is  the  secretion  of  the  preputial  gland — the  so-called 
smegma  prseputii  and  the  cerumen  of  the  ceruminous  glands  of  the 
external  ear.  In  addition  to  the  common  constituents  of  the  sebum, 
the  smegma  also  contains  certain  components  of  the  urine  and  their 
decomposition-products,  such  as  ammonium  soaps,  and  in  the  horse 
hippuric  acid,  benzoic  acid,  and  oxalate  of  lime.  Its  peculiar  odor 
in  man  is  no  doubt  due  to  the  presence  of  certain  fatty  acids.  In 
the  secretion  of  the  beaver — the  castoreum  of  the  shops — this  is 
thought  to  be  referable  to  a  phenol-like  body,  while  in  the  corre- 
sponding product  of  the  musk-deer  (muse)  a  volatile  base  is,  accord- 
ing to  Wohler,  the  active  odorous  principle. 

The  cerumen  differs  from  the  common  sebum  in  containing  a 
very  considerable  proportion  of  potassium  soaps.  In  addition,  we 
also  meet  with  a  peculiar  yellow  pigment,  which  has  an  exceedingly 
bitter  taste  ;  but  of  the  composition  of  this  nothing  is  known. 

In  the  skin  glands  of  certain  amphibia,  finally,  and  notably  the 
toad  and  the  salamander,  poisonous  substances  have  been  found 
which  in  their  physiological  effect  closely  resemble  the  action  of 
digitalis  and  strychnin.  They  have  been  termed  bufidin  and  sala- 
mandrin,  respectively.  In  the  secretion  of  the  toad,  moreover, 
methyl-carbylamin  and  isocyan-acetic  acid  have  been  found,  of  which 
the  former  is  especially  toxic. 


CHAPTER  XXI. 

THE  GLANDULAR  ORGANS. 

THE  LIVER. 

The  functions  of  the  liver,  as  is  apparent  from  a  survey  of  the 
foregoing  chapters,  are  manifold.  During  embryonic  life  the  organ 
is  intimately  concerned  in  the  production  of  red  corpuscles,  and  at 
this  time  already  manifests  its  function  as  an  excretory  organ  also 
in  the  production  of  bile.  After  birth  its  hsemapoietic  activity 
ceases,  but  it  continues  important  as  an  excretory  organ  through 
which  the  decomposition-products  of  haemoglobin,  in  so  far  as  they 
are  not  retained  and  utilized  in  the  formation  of  new  corpuscles,  are 
eliminated  from  the  body  in  association  with  taurin,  glycocoll, 
cholesterin,  and  the  cholalic  acids.  At  the  same  time,  however,  the 
liver  is  the  seat  of  some  of  the  most  important  syntheses  which  occur 
in  the  animal  body,  and  in  which  both  anabolic  and  katabolic  prod- 
ucts of  the  metabolism  are  involved.  We  have  thus  seen  that  the 
greater  portion  of  urea  in  mammals,  and  of  uric  acid  in  birds  and 
reptiles,  is  here  produced,  and  we  have  also  pointed  out  that  certain 
aromatic  substances  which  are  formed  during  the  process  of  intes- 
tinal putrefaction  or  have  been  ingested  as  such  are  transformed 
in  the  liver  into  conjugate  sulphates  and  glucuronates  and  are  thus 
rendered  innocuous.  Still  other  substances,  moreover,  which  are  for- 
eign to  the  body,  such  as  various  metallic  salts  and  certain  alkaloids, 
are  here  removed  from  the  general  circulation  when  artificially  intro- 
duced, and  it  is  for  this  reason  also  that  the  hypodermic  injection 
of  such  substances  is  much  more  efficacious  than  their  administra- 
tion by  the  mouth.  The  subsequent  elimination  of  the  metallic  salts 
then  occurs  in  part  through  the  bile,  and  to  a  great  extent  also 
through  the  intestinal  epithelium.  The  alkaloids  are  similarly  re- 
moved, and  also  appear  in  the  urine  in  a  more  or  less  modified  form. 

Formerly  it  was  supposed  that  the  retransformation  of  peptones 
into  native  albumins  occurred  in  the  liver,  but,  as  I  have  shown, 
tlii-  h  not  the  case.  On  the  other  hand,  we  have  seen  that  the  carbo- 
hydrates after  their  transformation  into  monosaccharides  arc  carried 
to  the  liver,  and  arc  here  stored  in  the  form  of  glycogen  when 
an  immediate  demand  for  glucose  does  not  exist  on  the  part  of  the 
other  organs  and  tissues  <>f  the  body.  This  transformation  of  mono- 
saccharides into  glycogen  represents  one  of  the  vaoai  important  syn- 
theses which  OCCUr  in  the  animal  body,  and  it  is  interesting  to  note 
that  whereas  glycogen  on  decomposition  always  gives  rise  to  the  for- 
mation of  glucose,  the  liver  is  capable  of  transforming  the  other 
monosaccharides  into  glycogen  as  well  (see  also  page  167.) 

399 


400  THE  GLANDULAR   ORGANS. 

Of  the  forces  which  are  at  work  in  bringing  about  these  various 
changes  in  the  liver  we  know  very  little,  but  to  judge  from  recent 
observations  it  appears  that  certain  tissue  ferments  are  here  primarily 
concerned. 

The  reaction  of  the  living  liver-tissue  is  alkaline.  After  death,, 
however,  it  becomes  acid,  and  there  is  reason  to  believe  that,  as  in 
the  case  of  the  muscle-tissue,  this  acid  reaction  is  essentially  refer- 
able to  the  formation  of  lactic  acid.  At  the  same  time  the  tissue 
becomes  opaque,  owing  to  a  coagulation  of  the  liver  albumins. 

A  general  idea  of  the  chemical  composition  of  the  liver  may  be 
formed  from  the  following  analyses,  which  are  taken  from  v.  Bibra : 

Man.  Ox. 

Water 761.7  713.9 

Solids 238.3  286.1 

Soluble  albumins 24.0               23.5 

Albuminoids 33.7              62.5 

Fats 25.0              328 

Extractives 60.7              49.1 

Insoluble  portion 94.4  112.9 

The  mineral  salts,  according  to  v.  Bibra,  constitute  about  1  per 
cent,  of  the  fresh  tissue,  and  are  essentially  represented  by  the  phos- 
phates of  potassium  and  sodium  and  a  fairly  large  amount  of  iron. 
Traces  of  manganese,  copper,  and  lead  are  also  found. 

The  Albumins. — The  albumins  of  the  liver-tissue  have  been 
notably  studied  by  Halliburton  and  Plosz.  The  gland  was  freed 
from  blood  and  bile  by  transfusion  with  ice-water  containing  0.75 
per  cent,  of  common  salt.  The  tissue  was  then  cut  into  small  pieces 
with  cooled  knives,  frozen  and  placed  under  pressure.  On  thawing, 
an  alkaline  fluid  could  thus  be  obtained,  which  represents  the  liver- 
plasma.  In  this  fluid  a  globulin  exists  which  coagulates  at  45°  C, 
and  is  regarded  by  Halliburton  as  being  possibly  identical  with  one 
of  his  cell-globulins.  It  can  be  digested  by  gastric  juice.  In  addi- 
tion a  nucleo-albumin  was  found,  which  coagulated  at  70°  C,  and 
which  yielded  an  insoluble  residue  of  nuclein  on  digestion.  From 
the  cells  proper  they  extracted  a  globulin  with  a  10  per  cent,  solution 
of  sodium  chloride,  which  coagulated  at  75°  O,  and  which  may  also 
be  identical  with  one  of  Halliburton's  cell-globulins ;  further,  an 
albumin  (coagulation -point  70°-73°  C.)  and  an  alkaline  albuminate. 
In  addition,  a  glucoproteid  has  also  been  demonstrated,  which  yields 
a  reducing  substance  on  boiling  with  dilute  mineral  acids,  and 
which  is  probably  of  a  mucinous  character  and  derived  from  the 
connective  tissue  of  the  organ. 

The  nuclei  finally  contain  nucleins,  and  it  is  of  special  interest  to 
note  that  at  least  two  of  these  contain  iron.  The  one  is  apparently 
identical  with,  or  at  least  closely  related  to,  the  hwmatogen  of  birds' 
eggs,  while  in  the  other,  which  Zaleski  terms  hepatin,  the  iron  is 
even  more  firmly  combined.  The  occurrence  of  these  iron-contain- 
ing nucleins  is  important  in  view  of  the  fact  that  the  iron  which 
is    furnished  in  the  food  can  apparently   be  utilized   only  by    the 


THE  LIVER.  401 

body  in  the  formation  of  haemoglobin,  when  introduced  in  such 
form.  It  has  hence  been  suggested  that  these  nucleins  after  resorp- 
tion are  temporarily  deposited  in  the  liver  until  required  by  the 
baemapoietic  organs. 

The  iron  which  is  present  in  the  liver  in  molecular  combination 
with  the  nucleins  can  be  demonstrated  only  after  the  isolation  of  the 
nucleins  in  question  and  their  incineration.  But  in  addition  we 
also  find  iron  in  combination  with  albumins  as  so-called  iron- 
albuminates,  from  which  the  metal  can  be  split  off  by  treating 
with  acid  alcohol.  The  presence  of  this  form  can  be  directly 
demonstrated  by  moistening  a  slice  of  the  liver-tissue  with  hydro- 
chloric acid,  and  then  with  a  solution  of  potassium  ferrocyanide  or 
p  itassium  sulphocyanide,  when  a  blue,  viz.,  a  red  color  develops. 
As  regards  the  origin  of  these  iron-albuminates,  the  opinion  prevails 
that  they  are  formed  within  the  tissues  of  the  body,  and  are  refer- 
able to  the  disintegration  of  red  corpuscles.  They  are  accordingly 
also  found  in  the  spleen  and  the  bone-marrow,  and  are  met  with 
in  increased  amounts  in  conditions  which  are  associated  with  an 
increased  destruction  of  red  corpuscles.  Large  quantities  are  thus 
especially  noted  in  cases  of  pernicious  anaemia,  in  acute  infantile 
gastritis,  and  in  poisoning  with  arsenious  hydride,  where  their 
presence  constitutes  the  phenomenon  of  so-called  siderosis  of  the 
liver.  According  to  Vay,  the  average  quantity  of  iron-albuminate 
which  can  be  isolated  from  the  fresh  organ  under  normal  conditions 
amounts  to  from  0.15  to  0.3  per  cent.,  corresponding  to  from  0.01 
to  0.018  per  cent,  of  iron. 

The  occurrence  of  especially  large  amounts  of  iron  in  the  liver  of 
newly  horn  animals  is  probably  referable  to  the  haemapoietic  activity 
of  the  organ  during  embryonic  life. 

Isolation  of  the  Iron-containing  Nucleins. — To  prevent  any  con- 
tamination with  haemoglobin,  it  is  necessary  to  remove  all  traces  of 
blood  from  the  liver.  To  this  ondy  Bunge  has  suggested  the  follow- 
ing method  :  in  the  living  animal  which  has  been  anaesthetized  with 
morphin  and  chloroform  a  cannula  is  tied  into  the  portal  vein. 
Through  this  a  stream  of  a  1  per  cent,  solution  of  sodium  chlo- 
ride heated  to  the  body  temperature  is  introduced  under  moderate 
pressure.  As  Boon  as  the  solution  begins  to  flow  the  hepatic  artery 
and  the  hepatic  veins  are  divided  and  the  abdomen  closed.  A 
minute  later  it  is  reopened,  the  liver  is  dissected  out  and  placed 
in  a  porcelain  bowl,  while  the  transfusion  is  continued.  The  bowls 
arc  changed  until  perfectly  clear  saline  solution  flows  from  the  veins. 
To  attain  thi-  end,  the  transfusion  need  l»e  carried  on  for  only  a  few 
minutes.  If  successfully  performed,  the  liver  should  present  a  uni- 
formly light-brown  color,  and  a  portion  of  the  minced  organ  when 
placed  in  distilled  water  should  leave  this  entirely  uncolored.  The 
gall-bladder  i-  now  removed,  the  organ  pressed  between  filter-paper, 
finely    bashed,  and  enveloped    in    muslin.     It   is  (hen    thoroughly 

kneaded    under  water.      The    connective   tissue  and    vessels  :ire  thus 
2fi 


402  THE  GLANDULAR   ORGANS. 

separated  from  the  cellular  elements  and  remain  behind.  The  cells 
are  thoroughly  extracted  with  water  and  with  dilute  saline  solution, 
by  decantation,  until  all  soluble  substances  have  been  removed. 
They  are  then  digested  with  gastric  juice.  The  non-digested  residue 
is  extracted  with  acidulated  alcohol  and  subsequently  with  ether,  to 
remove  pigments,  cholesterin,  and  fats.  It  is  then  treated  with 
weak  ammonia-water,  which  dissolves  the  iron-containing  nucleins. 
From  this  solution  they  are  precipitated  with  absolute  alcohol  when 
added  in  excess.  The  resulting  material  constitutes  the  hepatin  of 
Zaleski.  The  other  iron-containing  nuclein  is  apparently  present 
in  the  liver  as  a  nucleo-albumin,  and  is  found  in  this  form  in  the 
saline  extract  of  the  cells.  To  demonstrate  its  presence,  the  previ- 
ous extraction  with  saline  solution  is  omitted.  If  the  residue  of 
nucleins,  which  remains  after  digestion  with  gastric  juice,  is  then 
placed  in  a  solution  of  ammonium  sulphide,  a  greenish  color  gradu- 
ally develops  which  ultimately  turns  black,  owing  to  the  forma- 
tion of  sulphide  of  iron.  The  hepatin  itself  does  not  give  this 
reaction.  Neither  substance  gives  up  its  iron,  even  when  treated 
with  acidulated  alcohol1  for  days,  thus  differing  from  the  iron  albu- 
minate, which  behaves  in  this  manner  exactly  like  inorganic  prep- 
arations of  iron. 

Isolation  of  the  Iron- containing  Albuminates. — In  this  case  it  is 
not  necessary  previously  to  wash  out  the  blood.  The  organ  is 
minced  without  further  preparation,  and  is  placed  in  from  three  to 
four  times  its  volume  of  water.  The  mixture  is  slowly  heated, 
boiled  for  about  fifteen  minutes,  and  filtered  on  cooling.  The  filtrate 
is  carefully  precipitated  with  a  10  per  cent,  solution  of  tartaric  acid. 
The  resulting  flocculent  material,  which  presents  a  brown  color,  is 
collected  on  a  filter,  washed  with  a  weak  solution  of  tartaric  acid, 
then  with  50  per  cent,  alcohol,  and  finally  with  absolute  alcohol.  It 
contains  about  6  per  cent,  of  iron.  It  is  soluble  in  solutions  of  the 
alkalies,  and  does  not  react  with  ammonium  sulphide  at  once.  After 
a  few  minutes,  however,  the  solution  becomes  darker  and  gradually 
turns  black.  On  treating  with  acid  alcohol  (see  above)  the  iron  is 
split  off,  and  can  be  directly  demonstrated  by  testing  with  potassium 
ferrocyanide  or  potassium  sulphocyanide.  Schmiedeberg  has  termed 
the  substance  in  question  ferratin,  and  regards  it  as  a  ferri-albuminic 
acid. 

Ferments. — The  ferments  which  occur  in  the  liver  are  as  yet  but 
little  known.  It  appears  that  several  varieties  exist,  and  it  is  quite 
probable  that  they  are  intimately  concerned  in  the  various  functions 
of  the  organ.  Some  of  the  ferments  are  oxydases,  and  one  of  these 
in  turn  is  an  aldehydase,  viz.,  a  ferment  that  is  capable  of  oxidizing 
salicylic  aldehyde  to  the  corresponding  acid.  The  existence  of 
another  ferment  which  is  capable  of  transforming  firmly  combined 
nitrogen  into    amido-nitrogen   seems   to  have   been   established  by 

'The  acidulated  alcohol  contains  10  volumes  of  a  25  per  cent,  solution  of  hydro- 
chloric acid  and  90  volumes  of  96  per  cent,  alcohol  (Range's  fluid). 


THE  LIVER.  403 

Jacoby.  To  the  presence  of  this  latter  autolytic  phenomena,  which 
have  been  described  by  Salkowski  and  his  pupils,  are  possibly  due. 
A  urea-forming  enzyme  also  is  said  to  occur  in  the  liver,  but  its 
existence  has  not  as  yet  been  satisfactorily  demonstrated. 

Glycogen. — Amount. — The  amount  of  glycogen  which  occurs  in 
the  liver  is  primarily  dependent  upon  the  state  of  nutrition  of  the 
animal  and  the  amount  of  exercise  that  is  taken.  This  is  apparent 
from  the  fact  that  it  is  constantly  consumed  during  the  activity  of  the 
muscle-tissue  more  especially,  but  is  also  utilized  in  the  regeneration 
<>f  all  cellular  elements  of  the  body.  During  starvation  it  rapidly 
disappears,  but  it  is  also  rapidly  formed  if  carbohydrates  are  then 
ingested.  Maximal  amounts,  according  to  Kiilz,  are  found  after 
from  fourteen  to  sixteen  hours  following  the  administration  of  food. 
It  has  been  calculated  that  in  the  liver  of  man  150  grammes  can  be 
stored  at  one  time.  This  would  correspond  to  about  10  per  cent, 
for  an  or^an  weigdiino-  1500  grammes.  In  dogs  which  have  been 
fed  on  potatoes  and  bread  Pavy  claims  to  have  found  as  much  as  17 
per  cent.  After  death  the  transformation  of  glycogen  into  glucose 
continues  as  in  the  case  of  muscle-tissue,  and  in  order  to  ascertain 
the  exact  amount  which  was  present  during  life  it  is  hence  necessary 
to  remove  the  organ  at  once  and  to  prevent  the  further  inversion  of 
the  material,  by  the  living  protoplasm  or  the  contained  ferments,  by 
placing  the  tissue  in  boiling  water. 

Properties. — The  pure  substance  represents  a  white,  amorphous 
powder,  which  is  both  odorless  and  tasteless.  In  water  it  forms  an 
opalescent  solution,  from  which  it  can  be  precipitated  by  the  addi- 
tion of  alcohol,  after  adding  a  little  sodium  chloride,  or  by  means 
of  lead  subacetate.  The  substance  is  dextrorotatory.  The  specific 
degree  of  rotation,  however,  seems  to  be  influenced  by  various 
factor-.  In  pure  solution  it  is  given  as  f-  196.63°.  It  does  not 
reduce  Fehlin^'s  solution,  but  can  maintain  cupric  hydroxide  in 
solution.  After  the  addition  of  a  little  sodium  chloride  its  solutions 
an;  colored  red  by  treating  with  iodine.  With  benzoyl  chloride,  in 
the  presence  of  sodium  hydrate,  it  gives  a  granular  precipitate  of 
benzoyl-glycogen.  On  boiling  with  dilute  mineral  acids  it  is  trans- 
formed into  glucose.  Ferments  invert  it  to  maltose  or  glucose, 
according  to  the  nature  of  the  enzymes  at  work. 

Isolation  and  Quantitative  Estimation. — The  perfectly  fresh  liver, 
immediately  after  removal  from  the  animal,  is  placed  in  boiling 
water  and  divided  into  -mall  pieces.  After  boiling  for  a  few 
minute-,  these  are  removed,  ground  to  a  pulp  with  sand  or  pulverized 
glass,  and  then  boiled  in  a  1  per  cent,  solution  of  sodium  hydrate, 
using  100  c.c.  1  or  every  100  grammes  of  tissue.  With  liver-tissue 
two  to  three  hour-  suffice,  while  with  muscle-tissue  it  is  best  to  boil 

for  from  four  to  eighl   hour-.      Care  must  be  h;i<l  during  this  process 

thai  the  concentration  of  the  alkali  does  not  exceed  2  percent.;  to 
this  end  water  i-  added  from  time  to  time.     The  alkaline  extract 

after  filtration  i->  then  united  with  the  watery  solution  first  obtained, 


404  THE  GLANDULAR   ORGANS. 

and  neutralized  with  hydrochloric  acid.  After  concentrating  the 
resulting  solution,  the  remaining  albumins,  notably  gelatin,  are  pre- 
cipitated on  cooling  by  alternate  treatment  .with  a  solution  of  iodo- 
mercuric  iodide  and  hydrochloric  acid  added  drop  by  drop.  In 
the  filtrate  the  glycogen  is  precipitated  with  an  excess  of  alcohol. 
It  is  collected  on  a  filter,  washed  with  60  per  cent,  alcohol,  then 
with  absolute  alcohol  and  ether,  and  is  finally  dried  in  a  desic- 
cator over  sulphuric  acid.  From  the  weight  thus  obtained,  that  of 
the  combined  mineral  salts  must  be  deducted  after  incineration. 

Glucose. — The  amount  of  glucose  in  the  perfectly  fresh  liver 
varies  between  0.2  and  0.6  per  cent.,  but  rapidly  increases  at  the 
expense  of  the  glycogen  after  the  removal  of  the  organ  from  the 
body.  To  obtain  results  which  represent  the  actual  amount  that 
is  present  during  life,  it  is  hence  necessary  to  eliminate  the  inverting 
action  of  the  living  protoplasm  and  of  ferments  by  placing  the  organ 
in  boiling  water  immediately  after  the  death  of  the  animal.  It  is 
then  finely  minced,  thoroughly  extracted  with  boiling  water,  and  the 
sugar  determined  in  the  filtrate  according  to  the  usual  methods. 

Fat. — The  amount  of  fat  which  is  found  in  the  liver  is  quite 
large,  as  compared  with  the  other  organs  of  the  body,  and  normally 
varies  between  2  and  3.5  per  cent.  It  is  deposited  in  the  cells,  and 
beginning  along  the  periphery  of  the  acini  increases  in  amount  toward 
the  centre.  It  is  most  abundant  after  meals,  and  to  a  certain  degree  is 
dependent  upon  the  amount  of  fat  ingested.  Under  suitable  condi- 
tions the  infiltration  may  become  so  marked  as  to  simulate  fatty 
degeneration ;  but,  in  contradistinction  to  fatty  infiltration,  we  find 
that  in  fatty  degeneration  the  amount  of  the  solids  is  markedly 
diminished.  The  amount  of  water  in  fatty  infiltration  is  diminished, 
while  in  degenerative  changes  it  is  perhaps  slightly  increased.  These 
relations  are  exemplified  by  the  following  figures,  which  are  taken 
from  Hammarsten  : 


Water.                          Fat.                      ^SSd?* 

Normal  liver    .    . 

770    pro  mille     20-35    pro  mille  207-195  pro  mille. 

Fatty  defeneration 

816      "       "             87         "       "             97         "       " 

Fatty  infiltration 

616-621  "      "       195-240    "      "       184-145    "      " 

Extractives. — The  extractives  which  are  found  in  the  liver, 
aside  from  glycogen  and  glucose,  are  notably  xanthin-bases,  which 
are  derived  from  the  nuclei.  They  comprise  xanthin,  hypoxanthin, 
guanin,  and  adenin.  Conjointly  they  represent  about  4.52  pro  mille 
of  the  dried  tissue.  They  can  be  isolated,  according  to  the  method 
described  on  page  363.  In  addition  we  find  small  amounts  of  urea, 
uric  acid,  paralactic  acid,  and  jecorin.  Cystin  also  has  been  isolated 
from  the  normal  liver  of  a  horse  and  from  that  of  the  porpoise,  but 
it  is  questionable  whether  the  substance  can  actually  be  regarded  as 
a  normal  constituent  of  the  gland.  It  has  once  been  obtained  from 
the  liver  of  a  patient  who  during  life  had  eliminated  cystin  in  the 
urine.     Under  pathological  conditions,  and  especially  in  acute  yellow 


THE  LYMPH-GLANDS.  405 

atrophy,  large  quantities  of  paralactie  acid  have  been  found,  in 
addition  to  a  notable  amount  of  leucin  and  tyrosin.  In  amyloid 
degeneration  of  the  organ  chondroitin-sulphuric  aeid  has  been 
observed.  Biliary  pigments  are  normally  not  encountered  in  the 
liver-cells,  but  quite  commonly  they  stain  these  an  intense  yellow  in 
cases  of  obstructive  jaundice.  These  various  constituents  have  been 
studied  in  the  foregoing  chapters,  and  need  not  be  reconsidered  at 
this  place. 

THE   DIGESTIVE    GLANDS. 

Tin-  chemical  composition  of  the  digestive  glands,  viz.,  the  salivary 
glands,  the  pancreas,  and  the  glands  of  the  stomach  and  the  intes- 
tinal canal,  is  essentially  expressed  in  the  composition  of  their  specific 
secretions,  bearing  in  mind,  however,  that  the  various  ferments  exist 
in  the  cells  as  pro-enzymes.  The  mucin  which  is  furnished  by  the 
sublingual  and  submaxillary  glands  and  the  small  mucous  glands  of 
tin-  stomach  and  the  intestinal  tract  is  similarly  present  as  a  mucin- 
ogen.  Of  common  components,  we  find  a  certain  amount  of  mineral 
salts,  traces  of  the  common  albumins,  nucleo-albumins,  and  nucleinic 
bases,  and  in  the  pancreas,  in  addition,  free  fatty  acids,  small  amounts 
of  leucin.  tyrosin,  inosit,  and  paralactie  acid.  In  the  pancreas  a 
highly  complex  nucleo-glucoproteid  is  also  found,  which  yields  a 
somewhat  less  complex  substance  of  the  same  character,  together 
with  a  eoagulable  albumin,  when  the  fresh  gland  is  boiled  with 
water.  The  proteid  is  held  in  solution  owing  to  its  combination 
with  an  alkali,  but  is  precipitated  as  such  on  treating  with  a  dilute 
acid.  Its  elementary  analysis  has  given  the  following  results : 
carbon,  43  per  cent.;  hydrogen,  •"> ;  nitrogen,  0.7;  and  phosphorus, 
4.5.  In  addition,  the  substance  contains  a  considerable  amount 
of  iron.  On  digestion  with  gastric  juice  a  nuclein  remains  behind, 
which  i-  very  rich  in  phosphorus.  Among  the  decomposition- 
products  which  result  on  hydrolysis  with  boiling  hydrochloric  acid 
we  find  a  pentose  and  a  large  amount  of  nucleinic  bases,  among 
which  guanin  is  especially  abundant.  According  to  Bang,  a  nucleinic 
acid  can  be  isolated  from  the  proteid,  as  also  from  the  pancreas  di- 
rectly, which  he  terms  guanylic  <t<-i<l,:\<  guanin  is  the  only  nucleinic 
base,  thai  can  be  obtained  on  decomposition.  Its  composition  is: 
carbon.  34.18  percent.;  hydrogen,  4.4-*> ;  nitrogen  and  phosphorus, 
7.64,  which  would  correspond  to  the  formula  r.,,ir,fXl(lP.,017.  On 
decomposition  the  substance  yielded  at  least  .°>5  percent,  of  guanin, 
about  •';"  per  cent,  of  a  pentose  (calculated  as  glucose),  and  as  a  third 
product,  ammonia. 

THE  LYMPH-GLANDS. 

The  lymph-glands  comprise  the  lymph-glands  proper, the  thymus 
gland,  and  the  spleen.  Their  fibrous  framework,  as  T  have  pointed 
out  (page  383),  consists  essentially  of  reticulin,  but  also  contains 
fibres  oi  collagen  and  elastin.     The  composition  of  the  cellular  ele- 


406  THE  GLANDULAR   ORGANS. 

ments  of  the  glands  has  been  considered  in  the  section  on  the  Animal 
Cell  and  in  studying  the  leucocytes  of  the  blood  (pages  303  and 
322).  To  recapitulate  in  brief,  the  cells  contain  small  amounts  of 
albumin,  a  very  large  proportion  of  nucleo-histon,  besides  lecithins, 
fats,  cholesterins,  traces  of  glycogen,  succinic  acid,  and  larger  amounts 
of  nucleinic  bases,  among  which  adenin  predominates. 

In  the  spleen  we  also  meet  with  uric  acid,  and,  as  in  the  liver, 
with  iron-containing  nucleins  and  iron  albuminates,  which  may  be 
isolated  as  there  described.  In  addition,  small  amounts  of  inosit, 
jecorin,  and  cerebrosides  may  be  encountered.  Of  special  inter- 
est is  the  fact,  which  has  been  established  by  Gulewitscfh,  that 
arginin  is  a  normal  constituent  of  the  spleen.  All  these  bodies 
have  been  described  and  require  no  further  consideration  at  this 
place. 

THE   KIDNEYS. 

In  addition  to  the  common  albuminoids  which  enter  into  the  com- 
position of  the  supporting  tissue  of  the  kidneys,  we  find  the  common 
extractives,  viz.,  nucleinic  bases,  uric  acid,  urea,  leucin,  inosit,  gly- 
cogen, fats,  and  at  times  taurin.  All  these  substances,  however,  are 
present  in  only  small  amounts.  On  one  occasion,  in  the  ox,  cystin 
has  also  been  encountered,  but  it  is  questionable  whether  this  is  a 
constant  constituent  of  the  organs.  Of  albumins,  Halliburton  has 
isolated  a  globulin  and  a  nucleo-albumin,  with  coagulation-points  of 
52°  C.  and  63°  C,  respectively.  In  addition,  a  mucin-like  body 
has  been  found,  which  does  not  yield  a  reducing  substance,  however, 
on  boiling  with  mineral  acids,  and  which  is  probably  a  nucleo- 
albumin.  It  is  notably  found  in  the  papillary  portion  of  the  kidneys, 
while  the  other  nucleo-albumin  is  principally  met  with  in  the  cortical 
portion.     Serum-albumin  is  said  to  be  absent. 

THE   MAMMARY   GLANDS. 

The  chemical  composition  of  the  mammary  glands  has  not  been 
studied  in  detail.  We  know,  however,  that  the  protoplasm  of 
the  functionally  active  glands  is  rich  in  albumins,  and  it  appears 
that,  as  in  the  .case  of  the  pancreas,  a  very  complex  nucleo-gluco- 
proteid  is  here  also  present,  and  is  probably  intimately  concerned  in 
the  formation  of  two  of  the  most  important  constituents  of  the  milk, 
viz.,  the  casein  and  lactose.  It  may  be  obtained  in  solution  by  first 
washing  the  gland  thoroughly  in  water,  so  as  to  free  it  from  milk  ; 
it  is  then  extracted  with  a  0.5  pro  mille  solution  of  sodium  hydrate 
at  ordinary  temperatures.  Such  solutions  also  contain  the  common 
albumins,  and  represent  an  exceedingly  viscid,  stringy  fluid,  from 
which  the  proteid  in  question  can  be  precipitated  by  acidifying  care- 
fully with  dilute  acetic  acid.  On  boiling  with  dilute  acids  the  sub- 
stance is  decomposed  into  albumin,  phosphoric  acid,  and  a  reducing 
substance  of  unknown  composition.  On  digestion  with  gastric  juice 
it  yields  a  paranuclein. 


THE  MILK. 


407 


V<  in  the  case  of  the  pancreas,  the  substance  is  decomposed  by 
boiling  the  -land  with  water.  A  coagukble  albumin  and  a  nucleo- 
elucoproteid.  which  is  somewhat  less  complex  than  the  original  sub- 
stance thus  result.  From  this  solution  the  proteid  can  be  precipi- 
tated by  the  addition  of  a  dilute  acid.  Like  its  niother-substance, 
it  also  yields  a  reducing  substance  on  hydrolytic  decomposition. 
Of  the  rclati.-n  of  the  latter  to  lactose  nothing  is  known  but  it  is 
noteworthy  that  this  is  formed  on  standing  if  a  functionally  active 
u-land,  while  perfectly  fresh,  is  ground  to  a  pulp  and  kept  in  normal 
salt  solution  at  the  temperature  of  the  body.  An  intermediary  prod- 
uct is  then  also  apparently  formed,  which  is  of  a  colloid  nature  but 
not  identical  with  glycogen.  In  view  of  recent  researches,  which 
tend  to  show  that  the  reducing  group  which  is  present  in  the  gluco- 
proteids  is.  in  the  case  of  the  mucins  at  least,  not  a  true  carbohydrate, 
but  of  the  nature  of  chondroitin-sulphuric  acid  or  an  allied  substance, 
it  would  be  exceedingly  interesting  to  ascertain  whether  the  reduc- 
ing substance  in  the  case  of  the  mammary  nucleo-glucoproteid  also 
may  not  be  of  this  order.  . 

Of  other  constituents  of  the  gland,  we  find  various  xanthin- 
bases  and  in  the  functionally  actiye  organ  also  a  certain  amount  ot 
fat  which  is  present  in  the  form  of  globules  of  variable  size,  in  the 
bodies  of  the  cells.  .  ... 

The  specific  secretorv  product  of  the  mammary  glands  is  the  milk. 
The  Milk.— The  milk  is  the  specific  secretory  product  ot  the 
mammary  glands,  and  constitutes  the  natural  food  of  all  mammals 
in  the  early  stages  of  their  extra-uterine  existence.  It  contains  all 
those  food-stuffs  which  are  necessary  for  the  maintenance  ot  lite, 
viz  albumins,  carbohydrates,  and  fats.  The  nutrient  components 
of  the  milk,  however,  are  more  or  less  specific  of  the  secretion 
in  question,  and  are  not  found  elsewhere  in  the  body  as  such. 
They  arc  produced  in  the  -land  itself  from  the  common  constituents 
of  the  blood.  Am  ong  these  the  albumins  are  the  most  important, 
alHl  there  can  be  little  doubt  at  the  present  time  that  the  fits  of 
the  milk  also  are  largely  referable  to  this  source.  This  is  appar- 
enl  ,yom  thefacl  thai  in  the  bitch,  for  example,  the  amount  of  tai 
increases  with  an  increased  ingestion  of  meat  that  is  free  from  fats, 
while  it  is  diminished  when  the  animal  is  fed  on  fats  only.  #  I  lie 
80-called  milk-SUgar  also  is  apparently  derived  from  albumins,  as 
the  substance  continues  to  be  formed  although  no  carbohydrates  are 
ingested.  It-  amount,  however,  is  then  somewhat  smaller,  and 
increases  if  cane-sugar  or  starch  i-  added  to  the  diet. 

General  Characteristics.— Fresh  milk  is  an  opaque,  white,  yellow- 
ish-white, or  bluish-White  liquid,  of  a  somewhat  creamy  consistence, 
a  more  or  less  sweetish  taste,  and  an  insipid  odor  which  is  peculiar 
to  the  particular  animal  from  which  the  milk  has  been  obtain..!. 
On  microscopical  examination  it  is  seen  that  the  opacity  is  largely 
due  to  the  presence  of  fat-globules,  wind,  vary  from  0.002 14  to 
00046  ram.  in  diameter,  and  number  from  200,000  to  5,000,000 


408  THE  GLANDULAR   ORGANS. 

per  cbmm.j  with  an  average  of  about  1,050,000.  The  fat  is  thus 
present  in  a  state  of  fine  emulsion,  but,  in  contradistinction  to  other 
emulsions,  in  feebly  alkaline  media,  it  cannot  be  extracted  by  shak- 
ing with  ether  directly,  or  at  least  only  with  much  difficulty.  If,  on 
the  other  hand,  an  acid  or  a  caustic  alkali  is  previously  added  to  the 
milk,  this  is  readily  accomplished.  From  this  observation  it  has 
been  concluded  that  each  fat-globule  is  surrounded  by  an  albumi- 
nous membrane,  the  haptogenic  membrane  of  Ascherson,  which  is 
dissolved  by  acids  and  alkalies,  but  which  normally  prevents  the 
solvent  action  of  the  ether  upon  the  contained  fat.  Later  investi- 
gations have  rendered  this  view  improbable,  however,  and,  as  a 
matter  of  fact,  no  one  has  ever  succeeded  in  demonstrating  the  pres- 
ence of  a  special  membrane.  The  normal  resistance  to  the  action  of 
ether  is  now  explained  upon  the  assumption  that  each  globule  is 
surrounded  by  a  delicate  layer  of  albumin,  which  does  not  consti- 
tute a  true  membrane,  however,  but  is  formed  as  a  result  of  molecu- 
lar attraction.  It  is  possible,  indeed,  to  prepare  emulsions  of  fat 
artificially  by  shaking  with  albuminous  solutions,  which  in  their 
behavior  to  ether  are  quite  similar  to  milk.  As  regards  the  char- 
acter of  the  particular  albumin  which  forms  this  layer,  our  knowl- 
edge is  not  complete.  It  has  been  supposed  by  some  that  it  is 
formed  by  casein,  but  there  are  reasons  for  believing  that  the 
albumins  of  the  milk  in  general  may  here  be  concerned. 

On  standing,  the  greater  portion  of  the  fat  rises  to  the  surface  of 
the  milk  and  forms  its  cream.  On  beating  the  milk  for  some 
time,  the  individual  fat-globules  are  caused  to  coalesce,  and  sepa- 
rate out  as  a  semisolid  mass,  which  constitutes  the  butter.  The 
remaining  liquid  is  termed  buttermilk,  and  still  contains  a  consider- 
able amount  of  fat  which  has  remained  in  emulsion. 

Besides  the  fat-globules  the  milk  contains  also  innumerable  gran- 
ules of  calcium  phosphate  (probably  a  mixture  of  diphosphates  and 
triphosphates)  in  suspension,  which  are  visible  only  on  microscopical 
examination  and  are  said  to  number  about  4,000,000  per  cbmm. 
On  filtration  through  a  Chamberlain  filter,  under  pressure,  these 
remain  behind  together  with  the  fat.  But  we  then  also  find  that 
one  of  the  most  important  albuminous  constituents  of  the  milk, 
viz.,  casein,  which  is  found  in  combination  with  lime,  is  likewise  not 
present  in  solution,  and  is  thus  obtained  in  the  form  of  a  thin,  jelly- 
like material.  The  filtrate  constitutes  the  milk-serum  and  contains 
those  components  of  the  fluid  which  are  present  in  a  state  of  actual 
solution. 

Upon  the  addition  of  chymosin  to  fresh  milk,  at  the  temperature 
of  the  body,  it  coagulates  almost  at  once.  The  resulting  clot, 
which  constitutes  cheese,  then  contracts  and  a  yellowish  fluid  gradu- 
ally appears,  which  is  termed  sweet  whey.  During  this  process  the 
reaction  of  the  milk  is  not  changed.  A  similar  coagulation  is  noted 
when  fresh  milk  is  allowed  to  stand  exposed  to  the  air.  In  this  case, 
however,  the  reaction  of  the  whey  is  acid,  owing  to  the  formation  of 


THE  MILK.  409 

lactic  acid  from  lactose  in  consequence  of  the  activity  of  certain 
micro-organisms. 

Perfectly  fresh  milk  does  not  coagulate  on  boiling-,  but  it  will  be 
noted  that  a  skin  forms  on  the  surface  of  the  milk,  which  is  rapidly 
reformed  when  removed.  This  consists  of  coagulated  casein  in  com- 
bination with  mineral  salts,  and  especially  phosphates  of  calcium. 
Actual  coagulation  does  not  occur,  even  if  a  current  of  carbon  dioxide 
has  previously  been  passed  through  the  liquid.  If  the  milk  has 
stood  for  some  time,  however,  and  lactic  acid  fermentation  has  begun, 
a  tendency  to  coagulation  soon  becomes  manifest,  and  at  different  stages 
this  may  then  be  effected  by  boiling  after  saturation  with  carbon 
dioxide,  then  by  boiling  alone,  subsequently  on  treating  with  carbon 
dioxide  without  boiling  ;  and  finally,  as  I  have  stated,  it  occurs 
spontaneously.  Sterilization  of  the  milk,  with  the  subsequent  exclu- 
sion of  micro-organisms,  as  also  the  addition  of  preservatives,  such 
as  boric  acid,  salicylic  acid,  thymol,  etc.,  will  prevent  lactic  acid  fer- 
mentation, and  consequently  also  coagulation  referable  to  this  source. 

On  exposure  to  the  air,  milk  is  said  to  absorb  its  own  volume  of 
oxygen  within  three  days. 

Amount. — -The  amount  of  milk  furnished  in  the  twenty-four  hours 
is,  of  course,  different  in  different  animals.  It  is  largely  dependent 
upon  the  development  of  the  glands,  and  accordingly  is  most  abun- 
dant in  those  animals  in  which  by  artificial  selection  a  marked  hyper- 
trophy of  the  organs  has  been  produced.  Some  cowrs  may  thus  yield 
24  liters  of  milk  in  the  twenty-four  hours.  The  amount  is  further 
influenced  by  the  age,  as  also  by  the  character  of  the  diet,  the  amount 
of  liquid  ingested,  etc.  Especially  important  is  the  character  of  the 
diet,  ami  notably  the  amount  of  albuminous  food  that  is  ingested. 
Where  this  is  deficient  the  amount  of  milk  is  diminished,  while, 
•■  eteris  paribus,  larger  amounts  are  furnished  if  an  abundance  of 
albumins  is  ingested. 

Women  furnish  from  900  to  1000  grammes  on  an  average  during 
the  height  of  lactation  ;  loOO  grammes  probably  represent  the  maxi- 
mum output.  Good  cows  commonly  yield  from  b'  to  10  liters,  goats 
and  sheep  about  1  liter,  in  the  twenty-four  hours.  With  the  gradual 
cessation  of  lactation  and  the  coincident  atrophy  of  the  mammary 
glands  the  amount  decreases,  until  finally  the  secretion  is  arrested 
entirely.  In  women  and  cows  the  period  of  lactation  usually  lasts 
about  ten  months. 

Specific  Gravity. — The  specific  gravity  of  the  milk  is  largely  de- 
pendent upon  the  amounl  of  fal  present,  and  is  much  the  same 
in  different  animals.  Its  normal  variations  are  seen  in  the  accom- 
panying table  : 

Woman     1.028-1.034 

1.029-1.034 

1.030-1.034 

MP 1.037   1.040 

\  1.029  1.035 

Mara      1.028-1.034 

Bitch      1.034   1.040 


410  THE   GLANDULAR   ORGANS. 

Skimmed  milk  is,  of  course,  specifically  heavier  than  full  milk, 
and  a  higher  specific  gravity  is  accordingly  also  noted  in  milk  which 
is  poor  in  fat  than  in  rich  milk. 

Reaction. — Woman's  milk  and  that  of  most  herbivorous  animals 
is  uniformly  amphoteric,  owing  to  the  presence  of  diacid  and  mon- 
acid  phosphates  in  association  with  the  calcium  compound  of  casein. 
The  relative  values  of  the  acid  and  basic  components  in  cows'  milk 
and  human  milk  are  given  below  in  terms  of  decinormal  sodium 
hydrate  and  sulphuric  acid  solution.  The  figures  have  reference  to 
100  c.c.  of  milk  and  are  average  values  (Courant) : 

TySTaOH  i10H2S04         Ratio. 

Human  milk      10.8  c.c.  3.6  c  c.  3:1 

Cows'  milk 41.0  c.c.         19.5  c.c.         2:1 

Human  milk  is  thus  relatively  more  alkaline  than  cows'  milk,  but 
is  absolutely  both  less  alkaline  and  less  acid. 

Mares'  milk  is  alkaline  and  that  of  the  carnivorous  animals  acid. 

Chemical  Composition. — A  general  idea  of  the  chemical  composi- 
tion of  the  milk  of  different  animals  and  of  woman  may  be  formed 
from  the  following  analyses,  which  are  taken  from  Konig,  Gorup- 
Besanez,  Hoppe-Seyler,  and  others  : 

Human.  sCow. 

Water : 872.40-892.90  842.8-860.0 

Solids 108.00-127.60  140.0-157.2 

Albumins  (total)        16.13-36.91  33.0-43.2 

Albumin  (proper)      3.50-     9.91  1.2-    2.8 

Casein      12.80-  27.00  30.2-  42.0 

Fats      25.60-  43.20  40.0-  64.7 

Lactose 53.90-  60.90  43.4-  50.0 

Salts 1.650-  4.200  6.3-     7.1 

A  survey  of  this  table  thus  shows  that  human  milk  contains  a 
smaller  amount  of  albumins  and  fats  but  more  lactose  than  cows' 
milk. 

In  addition  to  the  above  components  the  milk  contains  traces  of 
urea,  kreatin,  kreatinin,  hypoxanthin,  cholesterin,  animal  gum,  and, 
curiously  enough,  citric  acid,  which  is  present  as  a  calcium  salt  to 
the  extent  of  from  0.18  to  0.25  per  cent.  Besides  these,  we  find  a 
small  amount  of  lecithins  and  a  yellow  lipochrome. 

Goat.  Sheep.  Mare.  Ass.  Dog.  Cat. 

Water 869.1  835.0  900.6  900.0  754.4  816.3 

Solids 130.9  165.0  99.4  100.0  245.6  183.7 

Albumins    .    .  36.9  57.4  1S.9  21.0  99.1  90.8 

Fats      ....  40.9  61.4  10.9  13.0  95.7  33.3 

Lactose    ...  445  39.6  66.5  63.0  31.9  49.1 

Salts     ....  8.6  6.Q  3.1  3.0  7.3  5.8 

Analysis  of  the  inorganic  components  of  human  milk  has  given 
the  following  results  (Bunge) :  the  figures  of  the  first  column  were 
obtained  at  a  time  when  but  little  sodium  chloride  was  ingested, 
while  those  of  the  second  column  were  gotten  while  the  woman 
ingested  30  grammes  a  day  (the  total  ash  is  calculated  as  1000  parts 
by  weight) : 


THE  MILK. 


411 


I.  II. 

Potassium  (K20) 0.780  0.703 

Sodium  (Xa.,U)      0.232  0.257 

Calcium   (OaO) 0.328  0.343 

Magnesium   (MgO) 0.064  0.065 

Iron  (Fe203) 0.004  0.006 

Pbospborie  acid  (,P205) °-478  °-469 

Chlorine  (CI) 0.438  0.445 

The  differences  which  exist  in  the  composition  of  full  milk,  as 
compared  with  skimmed  milk,  cream,  buttermilk,  and  whey,  are 
shown  below  : 

Full  milk        Shimmed         c  Buttermilk.        Whey. 

(cows).  milk.  J 

Water 871.7  906.6  655.1  902.7  932.4 

Solids 128.3  93.4  344.U  97.3  67.6 

Albumins     .    .  35.5  31.1  35.5  35.5  8.5 

Fats       ....  36.9  7.4  267.5  9.3  2.3 

Lactose      .    .    .  48.8  47.5  35.2  37.3  47.0 

Lactic  acid   .    .  none  none  none  3.4  3.3 

Salts 7.4  7.4  6.1  6.7  6.5 

Of  gases,  milk  contains  a  small  amount  of  oxygen  and  nitrogen, 
and  from  5.8  to  7.5  per  cent,  of  carbon  dioxide,  which  can  be 
removed  with  the  exhaust  pump. 

The  Albumins. — The  albumins  which  are  found  in  milk  are  casein, 
lactalbumin,  and  so-called  lactoglobulin,  which  is  probably  identical 
with  the  serum-globulin  of  the  blood-plasma.  Of  these,  casein  is 
the  most  abundant  and  the  most  important. 

Casein. — Casein  is  a  nudeo-albumin, and  has  the  character  of 
a  dibasic  acid.  In  the  dry  state  it  occurs  as  a  white  amorphous 
powder,  which  i-  almost  insoluble  in  water,  in  dilute  acids,  and  solu- 
tion- of  the  neutral  salts.  In  dilute  solutions  of  the  alkaline  hydrates 
and  in  lime-water  it  dissolves  with  ease,  at  the  same  time  forming 
gaits.  Such  solution- are  neutral  or  slightly  acid  in  reaction,  accord- 
ing to  the  amount  of  alkali  that  lias  been  added,  which  is  owing  to  the 
formation  of  neutral  or  acid  salts,  respectively.  When  triturated  in 
water  with  calcium  or  sodium  carbonate,  the  carbonates  are  decom- 
posed with  the  liberation  of  carbon  dioxide;  the  same  salts  are  then 
formed  a-  in  the  case  of  the  alkaline  hydrates.  Soldner  has  isolated 
two  calcium  salts  of  casein,  containing  1.55  and  2.36  per  cent,  of 
calcium  oxide;  according  to  Courant,  these  are  dicalcium  and 
tricalcium  casein,  respectively.  The  salt>  of  casein  with  the  alkalies 
and  alkaline  earths  are  readily  soluble  in  water,  even  in  the  absence 
of  neutral  -,-ilr-,  and  arc  hence  not  precipitated  on  dialysis.  On 
decomposition  with  dilute  acids  the  free  casein  is  obtained  again  in 
insoluble  form.  Suspended  in  water,  the  substance  is  coagulated  on 
boiling,  t  ud  <an  then  no  longer  be  dissolved  without  undergoing 
denaturization,  as  on  boiling  with  acids  ami  alkalies.  Solutions  01 
the  casein  salts,  on  the  other  hand,  do  not  coagulate  on  boiling,  but 
form  a  surface  -kin,  :i-  in  the  case  of  milk.  The  salts  can  be  pre- 
cipitated  from   their   solutions  by  salting  with  sodium  chloride  or 


412  THE  GLANDULAR   ORGANS. 

magnesium  sulphate  to  saturation.  Metallic  salts,  such  as  copper 
sulphate,  also  precipitate  a  neutral  solution  completely. 

In  the  milk  the  casein  exists  as  a  neutral  lime  salt,  but  does  not 
occur  in  a  state  of  actual  solution,  as  has  been  pointed  out.  On 
filtering  milk  through  a  Chamberlain  filter  under  pressure  it 
remains  behind,  together  with  the  fat  and  calcium  phosphate,  as 
a  jelly-like  material.  On  treating  milk  with  a  dilute  acid  the 
casein  is  precipitated,  as  in  the  case  of  the  aqueous  solution  of  its 
salts.  To  a  certain  extent  this  may  occur  in  the  stomach,  providing 
that  a  sufficient  amount  of  free  hydrochloric  acid  is  present ;  but,  as 
we  have  seen,  the  gastric  juice  is  further  capable  of  effecting  the 
coagulation  of  lime-casein  even  though  hydrochloric  acid  is  absent. 
This  is  brought  about  through  the  specific  activity  of  the  milk- 
curdling  ferment  (chymosin) ;  but  it  is  to  be  noted  that  the  coagula- 
tion of  milk  is  in  this  case  not  directly  comparable  to  the  action  of 
an  acid ;  for  while  the  latter  merely  brings  about  the  separation  of 
the  casein  by  the  removal  of  its  basic  component,  Hammarsten  has 
shown  that  the  chymosin  previously  causes  a  partial  decomposition 
of  the  lime-casein  by  hydrolysis.  As  a  result,  a  small  amount  of  an 
albumose-like  substance  is  split  off,  which  is  found  in  the  whey, 
while  the  greater  portion  of  the  lime-casein  is  transformed  into  so- 
called  lime-para  casein.  The  paracasein  is  likewise  a  nucleo-albumin 
with  acid  properties,  and  forms  salts  with  the  alkalies  and  lime, 
which,  like  those  of  casein,  are  readily  soluble  in  water.  These 
salts  are  then  further  apt  to  combine  with  soluble  calcium  salts 
to  form  double  salts,  which  are  insoluble  in  nearly  neutral  solu- 
tions. As  the  milk  is  nearly  neutral  in  reaction,  and  as  soluble 
calcium  salts  are  at  the  same  time  present,  coagulation  consequently 
occurs.  The  resulting  clot  constitutes  what  is  commonly  known  as 
cheese. 

In  the  absence  of  soluble  calcium  salts  coagulation  does  not 
occur  after  addition  of  the  chymosin.  Lime-paracasein,  however,  is 
manifestly  formed,  as  upon  subsequent  treatment  with  a  soluble 
calcium  salt  the  fluid  coagulates  in  the  usual  manner.  That  the 
ferment  takes  no  part  in  the  process  of  coagulation  itself,  but  merely 
prepares  the  lime-casein,  as  it  were,  for  this  end,  can  readily  be 
demonstrated  by  boiling  the  solution  of  chymosin  and  lime-casein 
after  having  been  kept  at  a  temperature  of  about  35°  C.  for  a  few 
minutes.  The  ferment  is  thus  destroyed,  but  coagulation  occurs 
nevertheless  if  a  soluble  calcium  salt  is  now  added. 

The  pepsin  of  the  gastric  juice  plays  no  part  whatever  in  the 
coagulation  of  the  milk.  But  after  this  has  taken  place  the  actual 
digestion  of  the  precipitated  lime-paracasein  begins.  As  I  have 
pointed  out,  this  is  then  decomposed,  with  the  formation  of  a  para- 
nuclein  and  albumin,  which  latter  is  digested  in  the  normal  manner. 
A  hetero-albumose,  however,  is  not  formed  during  the  process. 

From  the  above  considerations  it  is  clear  that  all  those  factors 
which  tend  to  increase  the  amount  of  soluble  lime  salts  in  the  milk 


THE  MILK.  41o 

will  increase  the  tendency  of  the  lime-casein  to  coagulate  upon  the 
subsequent  addition  of  chymosin,  while  this  is  diminished  if  the 
soluble  salts  are  transformed  into  the  insoluble  form  or  if  their 
amount  is  diminished.  It  is  for  this  reason  also  that  boiled  milk 
does  not  coagulate  so  rapidly  as  fresh  milk,  as  the  free  carbonic  acid, 
which  holds  a  certain  amount  of  calcium  in  solution,  is  thereby 
removed.  The  common  addition  of  lime-water  to  milk  similarly 
increases  the  tendency  to  coagulation,  but  does  not  render  it  more 
digestible,  as  is  generally  supposed. 

The  coagulation  of  the  milk  which  occurs  spontaneously  on 
standing  is  analogous  to  that  which  results  upon  the  addition  of  a 
mineral  acid,  and  is  referable  to  the  formation  of  lactic  acid  from 
lactose  as  a  result  of  bacterial  action.  The  phenomenon  has 
nothing  in  common  with  the  coagulation  which  results  from 
chymosin,  and  is  merely  the  outcome  of  the  withdrawal  of  the  lime 
salts  from  the  lime-casein  and  the  liberation  of  the  latter. 

From  the  fact  that  the  coagulum  which  results  in  cows'  milk 
upon  the  addition  of  chymosin  is  much  tougher  and  denser  than 
that  which  is  obtained  with  human  milk,  it  has  been  concluded  that 
the  casein  of  the  two  is  not  identical.  Soxhlet,  however,  has  shown 
that  the  density  of  the  coagulum  is  primarily  dependent  upon  the 
concentration  of  the  casein  solution  and  the  amount  of  soluble  cal- 
cium salts  and  acid  phosphates  present.  As  this  is  much  greater  in 
cows'  milk  than  in  human  milk,  it  follows  that  marked  differences 
must  thus  exist.  There  is  evidence  to  show,  nevertheless,  that 
different  forms  of  casein  occur.  Elementary  analysis  of  human 
casein  (Hammarsten)  and  cows'  casein  (Wroblewski)  has  given 
the  following   results: 

Human,  0,  52.96 ;    H,  7.05;   N,  15.65  ;   S,  0.75;   P,  0.84  ;   O,  22.78  per  cent. 
Cows',     0,52.24;    11,7.32;   N,  14.97  ;    S,  1.11 ;    P,  0.68 ;   0,23.66     "      " 

The  difference  is  here  especially  noticeable  in  the  amount  of  sul- 
phur. Human  casein,  moreover,  is  not  so  readily  precipitated  by 
salting  or  by  the  addition  of  acids,  and  does  not  always  coagulate 
with  chymosin.  The  gastric  juice,  it  is  true,  can  precipitate  the 
substance,  but  it  readily  dissolves  in  an  excess  without  leaving  any 
residue  of  auclein.  From  this  observation  Szontagh  has  concluded 
thai  human  casein  is  in  reality  no  nuclco-albumin.  Bui  aside  from 
these  data  we  have  abundant  evidence  that  human  casein  and  cows' 
casein  are  not  identical,  in  the  fact  that  no  modification  of  cows' 
milk,  however  produced,  i-  so  readily  digested  by  the  infant  as  is 
human  milk. 

Like  all  albumins,  casein  is  optically  active;  its  specific-  rotation 
in  neutral  solution  is  —  ho  degrees. 

The  isolation  of  the  casein  from    milk  will    be  described  below,  in 

association  with  the  isolation  of  the  soluble  albumins.     These,  as   I 
have  already  said,  are  lactalbumin  and  lactoglobulin. 

LACTALBUMIN. —  Lactalbumin  i-  found   both  in  human   milk  and 


414  THE  GLANDULAR  ORGANS. 

cows'  milk,  and  is  manifestly  closely  related  to  the  common  serum- 
albumin  of  the  blood-plasma.  Its  specific  rotation,  however,  is 
markedly  less,  viz., — 37  degrees,  as  compared  with  — 62.6  to — 64.6 
degrees.  Its  composition  according  to  Sebelien  is  C,  52.19  per  cent. ; 
H,  7.18  ;  N.  15.77  ;  S,  1.73  j  and  O,  23.13  ;  while  that  of  serum- 
albumin  is  given  as  C,  52.25-53.06  per  cent. ;  II,  6.65-6.85 ;  N, 
15.88-16.04;  S,   1.8-2.25;  and    O,    22.25-22.97  (Hammarsten). 

Lactoglobulin. — The  lactoglobulin  which  has  been  isolated 
from  cows'  milk  seems  to  be  identical  with  the  serum-globulin 
of  the   blood.     It  requires  no  further  description. 

That  still  other  albuminous  substances  may  occur  in  the  milk  is 
possible ;  but  if  so,  they  are  present  only  in  traces  and  have  not  as 
yet  been  identified.  Albumoses  and  peptones  are  not  found  in 
fresh  milk.  According  to  Siegfried,  a  phosphor-earn ic  acid  can  be 
isolated  from  milk  after  removal  of  the  casein  and  the  coagulable 
albumins  ;  this,  however,  is  supposedly  not  identical  with  that  found 
in  muscle-plasma. 

Origin  of  the  Albumins. — Casein,  as  has  been  stated,  is  a  specific 
product  of  the  activity  of  the  mammary  glands,  and  is  probably 
formed  from  the  complex  nucleo-glucoproteid  which  occurs  in  the 
functionally  active  organ.  As  this  is  not  found  in  the  milk,  we  may 
conclude  that  after  its  formation  it  is  decomposed  and  probably 
yields  casein,  on  the  one  hand ;  while  its  reducing  radicle  may  be 
concerned  in  the  production  of  lactose. 

Of  the  origin  of  lactoglobulin  and  lactalbumin,  nothing  is  known ; 
but,  as  I  have  said,  the  former  is  probably  identical  with  the  serum- 
globulin  of  the  blood,  while  in  the  case  of  the  latter  we  may 
imagine  that  it  has  originated  through  a  peculiar  transformation  of 
the  serum-albumin. 

Isolation  of  the  Albumins  of  the  Milk. — Isolation  of  Casein. — 
The  milk  is  diluted  with  four  times  its  volume  of  water  and  acidi- 
fied with  acetic  acid  to  the  extent  of  0.75-1.0  pro  mille.  On  stand- 
ing, the  casein  separates  out  and  is  filtered  off.  It  is  purified  by 
repeated  solution  in  Avater  with  the  aid  of  a  little  caustic  alkali, 
filtration,  and  reprecipitation  with  acetic  acid.  It  is  then  washed 
with  water  and  freed  from  traces  of  fat  by  means  of  ether-alcohol. 
The  greater  portion  of  the  fat  remains  on  the  first  filter. 

Isolation  of  Lactoglobulin. — The  milk  is  saturated  with 
common  salt  in  substance,  which  precipitates  the  lime-casein  to- 
gether with  a  small  portion  of  the  globulin.  If  then  the  neutral 
filtrate  is  saturated  with  magnesium  sulphate  at  30°  C.,the  remain- 
ing portion  of  the  lactoglobulin  is  obtained.  This  is  purified  as 
described  on  page  314. 

Isolation  of  Lactalbumin. — The  lime-casein  and  globulin  are 
first  precipitated  by  salting  with  magnesium  sulphate  in  substance  at 
30°  C.,  and  are  filtered  off.  In  the  filtrate  the  lactalbumin  can 
then  be  demonstrated  by  acidifying  with  acetic  acid  to  the  extent 
of  a  little  less  than  1  per  cent.,  or  by  salting  with  ammonium  sul- 


THE  MILK.  415 

phate  or  sodium  sulphate  in  substance.  The  albumin  is  filtered  off 
and  purified  as  described  on  page  316. 

Quantitative  Estimation  of  the  Total  Albumins. — To  this  end,  a  few 
grammes  of  milk  are  diluted  with  water,  treated  with  a  small 
amount  of  sodium  chloride  solution,  and  precipitated  with  tannic 
acid  or  phosphotungstic  acid  in  excess.  In  the  precipitate,  which  is 
washed  with  water,  the  amount  of  nitrogen  is  then  estimated  by 
Kjeldahl's  method.  By  multiplying  the  result  by  6.37  in  the  case 
of  cows'  milk,  or  by  6.34  with  human  milk,  the  corresponding 
amount  of  albumin  in  ascertained.  The  nitrogen  of  some  of  the 
extractives  is  included  in  the  result,  but  may  be  ignored.  In  cows' 
milk  it  represents  about  one-sixteenth  of  the  total  amount  of  nitro- 
gen, and  in  human  milk  about  one-eleventh. 

Separate  Estimation  of  the  Casein  and  the  Soluble  Albumins. — A 
few  grammes  of  milk  are  diluted  with  two  or  three  volumes  of  a 
saturated  solution  of  magnesium  sulphate,  and  are  then  saturated 
with  the  salt  in  substance.  In  the  precipitate,  which  is  washed  with 
a  saturated  solution  of  the  salt,  the  nitrogen  is  then  determined  as 
above.  The  result  multiplied  by  6.37  indicates  the  amount  of 
casein.  The  amount  of  lactalbumin  can  be  ascertained  by  deduct- 
ing the  value  found  for  casein  from  the  total  amount  of  albumin,  or 
by  diluting  the  filtrate,  after  separation  of  the  casein,  precipitating 
with  tannic  acid,  and  determining  the  amount  of  nitrogen  as  before. 
In  this  case  also  we  multiply  by  6.37. 

The  results  for  casein  thus  obtained  are  not  absolutely  accurate, 
as  the  globulin  is  likewise  precipitated  by  magnesium  sulphate. 
Its  amount,  however,  is  so  small   that  it  may  well  be  disregarded. 

The  Fats. — The  fats  which  are  found  in  the  milk,  viz.,  in  butter, 
are  essentially  the  same  as  those  which  occur  elsewhere  in  the 
animal  body,  viz.,  stearin,  palmitin,  and  olein.  In  addition,  how- 
ever, we  also  find  small  amounts  of  the  triglycerides  of  myristinic 
acid,  butyric  acid,  and  capronic  acid,  and  traces  of  caprylic  acid, 
caprinic  acid,  laurinic  acid,  and  arachinic  acid. 

Stearin,  palmitin,  and  olein  constitute  about  98  per  cent,  of  the 
total  amount,  and  of  these,  olein  represents  about  29.4-39.2  per 
cent.  As  a  consequence  of  the  large  quantity  of  olein  which  is 
thus  present,  the  melting-point  of  butter  is  relatively  low,  viz., 
31'  -31°  C., while  it  solidifies  between  19°  and  24°  C.  ' 

In  addition  to  the  neutral  fiits,  butler  also  contains  about  7  per 
cent,  of  volatile  fatty  acids,  of  which  3.7—5.3  per  cent,  are  repre- 
sented by  butyric  acid  and  2—3.3  per  cent,  by  capronic  acid. 

Formic  acid  has  been  found    in  butter  which  had   been  exposed  to 

sunlight. 

Of  the  origin  of  the  fats  which  are    found    in    milk  we    know  that 

they  are  to  a  large  extent  derived  from  albumins,  and  I  have  already 
pointed  out  thai  their  amount  increases  with  a  diet  that  is  rich  in 
such  material,  even  though  no  fat  is  ingested  :it  nil.     They  diminish 


416  THE  GLANDULAR   ORGANS. 

materially  if  fat  alone  is  ingested,  and  are  not  increased  if  much  fat 
is  administered,  while  the  ingestion  of  albumin  remains  constant. 
They  are  probably  formed  in  the  gland  directly,  and  on  micro- 
scopical examination  it  is  possible  to  demonstrate  their  presence  in 
the  cells,  in  the  form  of  fine  globules,  which  are  soluble  in  ether, 
and  are  colored  black  on  treating  with  osmic  acid. 

To  isolate  the  individual  acids  which  enter  into  the  composition 
of  the  neutral  fats,  the  butter  is  first  saponified,  when  the  resulting 
soaps  may  be  separated  from  each  other  according  to  the  usual 
methods  of  analysis. 

Quantitative  Estimation. — The  amount  of  fat  in  milk  is  most 
conveniently  estimated  densimetrically  with  Soxhlet's  apparatus. 
To  this  end,  a  known  amount  of  milk  is  mixed  with  a  solution 
of  sodium  hydrate  and  the  fat  extracted  with  a  definite  quantity 
of  ether.  The  ethereal  solution  is  allowed  to  separate,  and  is 
then  forced  into  a  glass  cylinder  provided  with  an  aerometer.  From 
the  specific  gravity  the  percentage  of  fat  is  then  read  oif  from  a  table 
which  accompanies  the  apparatus.  The  latter  is  so  constructed  that 
evaporation  of  the  ether  cannot  occur. 

In  the  absence  of  such  an  apparatus  the  amount  of  fat  can  be 
ascertained  gravimetrically  as  follows  :  20  c.c.  of  milk  are  treated 
with  a  small  amount  of  sodium  hydrate  solution,  and  are  extracted 
with  80  c.c.  of  ether  which  has  been  saturated  with  water.  This  is 
clone  by  shaking  in  a  tightly  closed  bottle.  After  the  ethereal  ex- 
tract has  entirely  separated,  60  c.c.  are  placed  in  a  weighed  beaker  ; 
the  ether  is  allowed  to  evaporate  ;  the  residue  is  dried  and  weighed. 
The  result  is  calculated  out  for  80  c.c.  of  the  ethereal  extract, 
corresponding  to  20  c.c.  of  milk. 

Lactose. — In  the  animal  body  lactose  is  found  only  in  the  milk,  if 
we  disregard  the  small  amounts  that  may  appear  in  the  urine  of  nurs- 
ing females,  and  which  must  hence  of  necessity  occur  also  in  the 
blood.  It  is  formed  in  the  mammary  glands,  and  may  possibly 
be  related  to  the  reducing  substance  wliich  results  from  the  nucleo- 
glucoproteid  when  this  is  boiled  with  mineral  acids.  On  exposure 
to  the  air  it  undergoes  a  peculiar  fermentation,  with  the  formation 
of  lactic  acid.  This,  in  turn,  combines  with  the  calcium  of  the 
lime-casein,  and  as  a  result  the  casein  separates  out,  and  constitutes 
what  is  popularly  termed  clabber.  On  further  standing,  this  con- 
tracts, and  finally  floats  in  a  clear,  light-yellow  fluid — the  acid  whey. 
The  fermentation  in  question  is  produced  by  definite  micro-organ- 
isms, of  which  fourteen  varieties  are  now  known. 

On  inversion,  lactose  is  decomposed  into  glucose  and  galactose 
(see  page  58). 

Isolation. — To  isolate  lactose  from  milk,  this  is  first  curdled  by 
the  addition  of  chymosin.  The  filtrate  is  slightly  acidified  with 
acetic  acid  and  boiled,  so  as  to  remove  the  coagulable  albumins.  The 
second  filtrate  is  then  concentrated  to  a  small  volume,  when  on  cool- 


THE  COLOSTRUM.  417 

ing  the  lactose  crystallizes  out.  To  purify  the  substance,  this  is  dis- 
solved in  water,  decolorized  with  animal  charcoal,  and  recrystallized 
by  evaporation.  It  is  thus  obtained  in  the  form  of  white  rhombic 
prism-,  which  are  soluble  in  water,  but  insoluble  in  absolute  alcohol. 
The  substance  has  a  somewhat  sweetish  taste,  and  contains  one  mole- 
cule of  water  of  crystallization,  which  rapidly  escapes  at  130°  C. 

Estimation. — To  estimate  the  amount  of  lactose,  the  milk  must 
first  be  freed  from  fits  and  albumins.  To  this  end,  it  is  most  con- 
venient to  dilute  with  water  and  to  remove  the  casein  by  the  cau- 
tious addition  of  acetic  acid.  The  resulting  precipitate,  which 
contains  both  the  casei'n  and  the  fat,  is  filtered  off  and  the  filtrate 
boiled.  After  the  removal  of  the  precipitated  coagulablc  albumins, 
tli.'  sugar  is  then  estimated  in  the  filtrate  by  titrating  with  Knapp's 
solution,  as  described  in  the  section  on  the  Urine.  Ten  c.c.  of  the 
reagent  correspond  to  0.031  gramme  of  lactose,  providing  that  the 
solution  contains  fnnn  0.5  to  1  per  cent,  of  sugar. 

In  addition  to  lactose,  the  milk  contains  also  small  amounts  of  a 
reducing  substance,  which  is  supposedly  identical  with  Landwehr's 
animal  gum.  It  is  possible,  however,  that,  as  in  the  case  of  the 
reducing  substances  of  the  mucins  and  mucoids,  chondroitin-sul- 
phuric  acid  or  an  allied  substance  may  be  responsible  for  the 
reactions. 

Extractives. — Among  the  extractives  of  the  milk,  which  com- 
prise traces  of  urea,  kreatin,  kreatinin,  xanthin-bases,  lecithins, 
cholesterin,  and  citric  acid,  the  latter  is  of  especial  interest,  as  it 
is  apparently  also  formed  in  the  mammary  glands,  and  is  not  refer- 
able to  the  ingestion  of  the  substance  as  such.  It  has  been  found  in 
human  milk  as  well  as  in  cows'  milk,  and  is  notably  present  in  com- 
bination with  calcium.  Its  amount  in  cows'  milk  is  given  as  0.25 
per  cent.,  while  in  human  milk  a  somewhat  smaller  quantity  occurs. 

The  formula  of  the  acid  is  CH2.COOH.C(OH).COOH.CH2. 
OOOH,  viz.,  (',.I1.()7  ;  it  is  thus  oxy-propon-trioarbonic  acid. 

Colostrum. 

The  term  colostrum  is  applied  to  the  secretion  of  the  mammary 
glands  which  i-  furnished  by  the  female  animal  during  the  first 
days  of  lactation,  and  which  may  also  be  expressed  from  the  glands 
during  a  variable  period  preceding  parturition. 

On  microscopical  examination  such  fluid  is  seen  to  contain  innum- 
erable fat-globules,  and  in  addition  a  variable  number  of  granular 
cells,  which  are  capable  of  manifesting  amoeboid  movements.  These 
are  termed  colostrum-corpuscles,  and  are  commonly  regarded  as 
leucocytes.  This,  however,  is  doubtful.  According  to  Woodward, 
they  have  a  small  irregular,  but  much  degenerated  nucleus.  Of  the 
granules,  a  few  are  stained  by  osmic  acid,  while  none  of  them  takes 
up  either  acid,  neutral,  or  basic  dyes,  in  their  reactions  they  show 
the  characteristics  of  proteid  material. 

27 


418  THE  GLANDULAR   ORGANS. 

The  secretion  is  a  thick  yellowish  fluid,  of  an  alkaline  and  some- 
times acid  reaction,  and  a  specific  gravity  that  is  much  higher  than 
that  of  true  milk.  In  the  cow  this  varies  between  1.046  and 
1.080,  and  in  the  human  female  between  1.040  and  1.060.  This  is 
principally  owing  to  the  presence  of  large  amounts  of  lactalbumin 
and  lactoglobulin.  As  a  consequence,  the  colostrum  coagulates 
on  boiling,  while  true  milk,  as  we  have  seen,  is  then  covered  merely 
by  a  skin,  which  is  composed  of  casein  and  calcium  phosphates.  The 
total  quantity  of  the  coagulable  albumins  may  reach  15  per  cent., 
while  in  milk  about  0.5  per  cent,  is  the  rule. 

The  amount  of  casein  and  of  mineral  salts  in  colostrum  is  also 
somewhat  greater  than  in  milk,  and  it  is  further  stated  that  more 
lecithin  and  cholesterin  is  present.  The  quantity  of  fat  is  practi- 
cally the  same,  while  that  of  lactose  is  somewhat  smaller.  As  a 
result  of  the  increase  in  the  amount  of  albumins  and  of  mineral  salts, 
the  total  solids  are  also  proportionately  increased,  and  may  amount 
to  25.3  per  cent,  in  cows'  colostrum,  as  compared  with  12.8  per  cent, 
in  the  case  of  the  milk. 

The  quantitative  composition  of  the  colostrum  after  parturition  is 
rapidly  altered,  so  that  after  a  few  days  already  the  normal  compo- 
sition of  true  milk  is  approached.  This  is  well  shown  in  the  follow- 
ing table,  which  is  taken  from  Gautier.  The  results  have  reference 
to  the  human  being  and  the  cow,  and  are  expressed  in  percentages : 

Hitman  Being. 

Nine  days  be-    Day  of  partu-    Twenty-four  Nine  days  later, 
fore    partu-        rition.  hours  after 

rition.  parturition. 

Water 85.86  82.80  84.30  88.580 

Solids 14.15  17.20  15.70  11.420 

caSn1"8. : " : :  8'07}       40°         •  •       3-690 

Fat     .    .    '.    '.    '.    '.    '.      2.35  5.00  .    .  3.530 

Lactose 3.63  7.00  .    .  4.300 

Salts  and  extractives     0.54  .    .  0.512  0.169 

Cow. 

Immediately        Twenty-four     Three    days       Average  of 
after  partu-  hours  later.  later.  30  analyses. 

rition. 

Water 73.07  82.3S  78.70  74.05 

Solids 26.93  17.62  21.30  25.95 

Albumins      ....  16.56  4.50  7.50  13.62 

Casein 2.65  4.50  7.30  4.66 

Fat 3.54  4.75  4  00  3.43 

Lactose 3.00  2.85  1.50  2.66 

Salts  and  extractives  1.18  1.02  1.00  1.58 

So-called  witch's  milk  is  the  fluid  which  can  be  expressed  from 
the  mammary  glands  of  both  sexes  immediately  after  birth.  Its 
qualitative  composition  is  the  same  as  that  of  milk.  Like  the  colos- 
trum, it  is  said  to  contain  colostrum-corpuscles.  According  to 
Schlossberger,  HaufT,  and  others,  it  contains  from  1.05  to  2.8  per 
cent,  of  albumin,  0.82  to  1.46  per  cent,  of  fat,  and  0.9  to  6  per 


THE  REPRODUCTIVE  GLANDS.  419 

cent,  of  lactose.     It  thus  contains  a  smaller  amount  of  water  than 
the  milk.     The  secretion  ceases  several  weeks  after  birth. 

Uterine  milk  is  a  fluid  which  can  be  obtained  from  the  uterine 
glands  of  ruminants  after  careful  separation  of  the  chorion  villi.  It 
has  the  appearance  of  cream,  and  is  morphologically  and  chemically 
quite  similar  to   colostrum. 

THE   REPRODUCTIVE    GLANDS. 

The  Testicles. — Of  the  chemical  composition  of  the  testicles  as 
such,  little  is  known.  Aside  from  the  albuminoids  which  enter  into  the 
construction  of  the  supporting  tissues  of  the  glands,  and  the  extrac- 
tives which  are  common  to  all  organs  of  the  body,  we  notably  meet 
with  albumins,  among  which  the  nucleins  are  especially  abundant. 
In  addition,  serum-albumin,  a  globulin,  and  a  substance  which 
apparently  belongs  to  the  hyalins,  have  been  encountered.  Of  mineral 
suits,  we  notably  meet  with  the  chlorides  of  sodium  and  potassium. 
The  reaction  of  the  glands  is  alkaline. 

The  Semen.— The  specific  product  of  the  functional  activity  of 
the  testicles  is  represented  by  the  semen,  and  notably  its  morpho- 
logical elements,  the  spermatozoa,  which  result  from  the  spermato- 
genetic  cells  through  a  complicated  process  of  metamorphosis,  in 
which  the  cell-nuclei  are  especially  concerned.  On  its  passage  to 
the  outside  the  testicular  fluid  is  mixed  with  the  secretions  of  the 
seminal  vesicles,  the  glands  of  Gowper,  and  notably  with  the  secre- 
tion of  the  prostate  gland. 

Recently  ejaculated  semen  is  a  markedly  viscid,  white  or  yellow- 
ish-white, opaque  fluid  of  the  appearance  of  milk,  in  which  micro- 
scopical examination  reveals  the  presence  of  innumerable  sperma- 
tozoa, and  a  few  hyalin  globules,  which  are  derived  from  the  seminal 
vesicles  \  further,  isolated  testicular  and  urethral  cells,  prostatic 
c  >rpU9cles,  and  cellular  bodies  enclosing  lecithin  granules,  besides  a 
large  number  of  free  granules,  which  arc  apparently  of  an  albumin- 
ous nature.  In  a  fresh  specimen  the  normal  spermatozoa  are  ac- 
tively motile,  and  continue  so  for  a  variable  length  of  time  if  evapo- 
ration is  prevented  Tin'  movements  are  in  all  likelihood  analogous 
to  those  of  the  cilia  of  certain  epithelial  elements  of  the  body,  and  are 
arrested  by  the  addition  of  water,  dilute  acids,  alcohol,  ether,  strongly 
alkaline  Solutions,  etc.  in  dilute  alkaline  solutions,  on  the  other 
hand,  ami  those  of  the  neutral  salts  they  continue  for  a  long  time. 

Semen  i-  heavier  than  water,  and  falls  to  the  bottom  as  a  jelly- 
like mass :  at  the  same  time  a  light  flocculent  precipitate  develops, 
which  consist*  of  the  so-called  fibrin  of  Eenle.  On  exposure  to  the 
air  it  is  apparently   coagulated,  but   later  becomes  Liquid,  as  before. 

It-  reaction  i-   neutral  or  slightly  alkaline. 

Testicular  Bemen,  in  contradistinction  to  that  which  has  been 
ejaculated,  is  said  to  be  odorless.  After  emission,  however,  an  odor 
develops  which  is  suggestive  of  glutin,  and  is  supposedly  referable 


420  THE   GLANDULAR   ORGANS. 

to  the  presence  of  an  alkaloidal  substance — spermin.  In  combina- 
tion with  phosphoric  acid,  this  is  found  in  the  secretion  of  the 
prostate  gland,  as  phosphate  of  spermin,  and  is  partly  decomposed 
on  exposure  to  the  air,  with  the  liberation  of  the  free  base  (see 
below). 

The  Spermatic  Liquid. — The  liquid  in  which  the  spermatozoa 
are  suspended  is  nearly  transparent.  It  contains  a  small  amount  of 
mucin  (?) ;  a  nucleo-albumin,  which  has  been  termed  spermatin,  and 
which  is  precipitated  by  acetic  acid,  but  is  readily  soluble  in  an 
excess  of  the  reagent;  also,  cerebrin  and  lecithins,  phosphate  of 
spermin,  and  various  mineral  salts,  among  which  sodium  chloride  and 
the  phosphates  of  the  alkaline  earths  predominate. 

Spermin. — Spermin,  as  stated  above,  occurs  in  the  spermatic  liquid 
in  combination  with  phosphoric  acid,  as  phosphate  of  spermin  ;  it 
is  viewed  as  ethylenimin,  C2H5N,  and  is  manifestly  closely  related  to 
the  diethylene  diamin  (piperazin)  of  Ladenburg  and  Abel. 

To  the  free  base  the  peculiar  odor  of  the  semen  is,  as  I  have  said, 
supposedly  due.  This  disappears  after  a  short  while,  owing  to 
a  polymerization  of  the  ethylenimin  to  diethylene  diamin  (piperazin) 
as  shown  in  the  equation  : 

/H  7NHV 

2Nf  CH2  =  C2HZ         >C2H4. 
\CH2  NNH/ 

The  phosphate  can  be  readily  obtained  in  crystalline  form  on  slow 
evaporation  of  the  semen,  but  may  also  separate  out  spontaneously 
on  standing  for  about  twenty-four  hours.  It  occurs  in  the  form  of 
hexagonal  pyramids,  which  appear  under  the  microscope  as  flat 
needles.  They  are  soluble  in  dilute  acids  and  alkalies,  as  also  in 
ammonia,  less  readily  so  in  hot  water,  and  are  insoluble  in  alcohol, 
ether,  and  chloroform.  These  crystals  are  known  as  Bottcher's 
spermin-cry state,  and  are  probably  identical  with-  the  so-called 
(Jliarcot-Leyden  crystals,  which  are  commonly  found  in  asthmatic 
sputa,  and  also  occur  in  the  blood  and  lymph-gland  of  leukemic 
patients.  They  have  likewise  been  observed  in  dried  egg-albumin 
and  in  anatomical  specimens  preserved  in  alcohol,  and  also  develop 
in  red  bone-marrow  that  has  been  exposed  to  the  air  for  a  few  days. 
Heated  to  100°  C,  the  crystals  turn  yellow  and  melt  near  170°  C, 
but  are  at  the  same  time  decomposed. 

To  isolate  the  free  base,  the  semen  is  extracted  with  alcohol  and 
then  with  dilute  sulphuric  acid.  If  the  acid  extract  is  then  treated 
with  baryta-water  and  evaporated  at  a  low  temperature,  the  free 
base  is  obtained.  It  can  be  precipitated  from  its  solution  by  treating 
with  auric  chloride,  platinum  chloride,  argentic  nitrate,  tannic  acid, 
phosphotungstic  acid,  etc. 

Spermin  has  attracted  much  attention  of  late,  owing  to  the 
stimulating  effect  which  the  substance  is  supposed  to  exert  upon  the 
oxidation  processes  of  the  body,  the  functions  of  the  central  nervous 


THE  REPRODUCTIVE  GLAXDS.  421 

system,  and  the  reproductive  organs.  It  represents  the  active  prin- 
ciple of  Brown-Sequard's  elixir. 

The  Spermatozoa. — Our  knowledge  of  the  chemical  composi- 
tion of  the  spermatozoa  has  been  greatly  extended  within  recent 
years  through  the  researches  of  Kossel  and  his  pupils,  preceded  by 
those  of  Miescher  and  Piccard.  These  observers  were  able  to  show 
that  in  certain  fishes,  such  as  the  salmon,  sturgeon,  pike,  shad, 
herring,  and  mackerel,  substances  can  be  isolated  from  the  mature 
spermatozoa,  which  apparently  represent  the  simplest  forms  of 
albumin,  and  are  now  collectively  termed  protamins.  Their  general 
characteristics  have  already  been  described  (page  69),  and  I  shall 
merely  recall  at  this  place  that,  according  to  Kossel,  a  protamin 
radicle  is  contained  in  all  albumins  and  represents  the  fundamental 
nucleus  of  the  albuminous  molecule.  These  protamins,  of  which 
several  varieties  are  described,  and  which  yield  the  hexon-bases  on 
hydrolytic  decomposition,  are  supposedly  combined  with  nucleinic 
acids  to  form  nucleins.  The  individual  nucleinic  bases  which 
further  enter  into  the  construction  of  the  nucleinic  acids  are  the 
common  forms,  which  are  also  found  elsewhere  in  the  animal  body. 
But  it  appears  that  the  spermatozoa  of  different  animals  do  not  con- 
tain all  forms.  In  the  case  of  the  salmon,  Miescher  and  Piccard  thus 
found  guanin  and  hypoxanthin,  while  from  the  semen  of  the  carp 
Kossel  obtained  adenin  and  hypoxanthin,  as  also  small  amounts  of 
xanthin,  but  no  guanin.  Inoko,  on  the  other  hand,  claims  to  have 
found  all  forms  in  the  semen  of  the  salmon,  boar,  and  ox,  but 
states  that  the  relative  amounts  of  the  individual  forms  are  not 
constant. 

Immature  spermatozoa  appai-cntly  contain  no  protamins  as  such, 
and  researches  must  be  undertaken  to  ascertain  whether  the 
results  which  have  thus  far  been  obtained  in  the  lower  forms  of 
animal  life  also  hold  good  for  the  higher  forms.  But  even  so,  it  is 
apparent  that  the  protamins  play  an  important  rdle  in  the  process  of 
reproduction,  and  a  key  may  thus  be  furnished  which  will  admit  of 
an  insight  into  the  chemical  basis  of  those  mysterious  morphological 
changes  which  find  their  expression  in  the  development  of  the 
ovum.  For  there  can  be  no  doubt  that  in  those  animals  in  which 
the  presence  of  protamins  has  been  established  in  the  spermatozoa 
they  represent  the  essential  reproductive  elements  on  the  part  of 
lie-  male.  This  suggests  itself  at  once  from  a  survey  of  the 
analysis  of  the  spermatozoa  of  the  salmon  as  given  by  Miescher: 
The  nucleins,  which  are  here  referred  to  are,  according  to  Kossel, 
nucleinic  acids  : 

Per  Cent. 

NucleiM 48.68 

Protamine  (salmin)       ii ' >.T * > 

Other  albumiiu 10.32 

!.«•<  ithina 7.47 

Cliolefterin      2:2\ 

Fata       4.53 


422  THE  GLANDULAR   ORGANS. 

The  albumins  referred  to  in  this  table  have  not  been  studied  in 
detail.  One  of  them,  according  to  Miescher,  contains  4  per  cent,  of 
sulphur.  In  addition,  the  spermatozoa  are  said  to  contain  a  cere- 
broside,  which  is  similar  to  cerebrin ;  also  a  very  considerable 
proportion  of  inorganic  salts,  which  are  essentially  represented  by 
phosphates. 

Detailed  analyses  of  the  spermatozoa  of  the  higher  animals  and  of 
man  are  not  yet  available. 

As  regards  the  composition  of  the  separate  parts  of  the  sperma- 
tozoa very  little  is  known,  bat  it  seems  that  the  protamins,  com- 
bined with  nucleinic  acids,  are  the  most  important  components  of 
the  head.  The  tails  are  dissolved  in  gastric  juice  on  prolonged 
digestion,  and  hence  probably  consist  of  albumins.  As  a  whole, 
the  spermatozoa  are  exceedingly  resistant  to  ordinary  solvents.  They 
are  soluble  in  boiling  solutions  of  the  caustic  alkalies,  while  in 
concentrated  sulphuric  acid,  nitric  acid,  acetic  acid,  and  boiling- 
solutions  of  sodium  carbonate  they  dissolve  only  in  part.  They 
are  likewise  resistant  to  putrefactive  changes,  and  can  be  obtained 
from  dried  semen,  with  the  preservation  of  their  natural  form, 
by  placing  the  material  in  a  1  per  cent,  solution  of  sodium  chloride. 

For  a  detailed  description  of  the  methods  which  are  employed  in 
the  isolation  of  the  individual  protamins,  I  must  refer  the  reader  to 
the  articles  of  Kossel,  Kurajeff,  and  others.  A  satisfactory  result 
may  be  expected  only  if  the  spermatozoa  are  mature,  but  even  then 
no  protamins  may  be  found,  as  I  have  already  indicated.  Working 
with  mature  testicles  of  the  sea-trout,  I  was  unable  to  obtain  a  body 
of  this  order. 

The  Ovaries. — Thus  far  a  study  of  the  chemical  composition 
of  the  ovaries  has  not  revealed  any  special  points  of  interest. 
In  addition  to  collagen  and  mucins,  which  enter  into  the  con- 
struction of  the  supporting  tissue  of  the  organs,  nucleins  and  true 
albumins  have  also  been  found,  and  are  probably  derived  from  the 
contained  ova  and  other  cellular  elements. 

The  most  important  constituents  of  the  cortex  of  the  gland,  viz.r 
the  Graafian  follicles,  which  enclose  the  specific  product  of  the  func- 
tional activity  of  the  ovaries,  viz.,  the  ova,  have  for  obvious  reasons 
not  been  open  to  a  detailed  investigation.  The  contained  fluid 
is  apparently  serous  in  character.  After  the  discharge  of  the 
ova  the  remaining  follicles  are  first  filled  with  blood  from  the  torn 
vessels  of  the  vesicle,  and  are  subsequently  transformed  into  the 
so-called  corpora  lutea.  The  yellow  color  of  these  is  owing  to  lipo- 
chromes,  or  luteins,  of  which  an  amorphous  and  a  crystalline  form 
may  be  isolated  (see  also  page  429). 

The  Ovum. — Of  the  chemical  composition  of  the  ova  of  the 
human  being  and  mammals  in  general,  nothing  definite  is  known, 
as  it  is  impossible  to  collect  them  for  purposes  of  analysis.  The 
eggs  of  fishes,  amphibia,  reptiles,  and  especially  of  birds,  on   the 


THE  REPRODUCTIVE  GLANDS.  423 

other  hand,  can  readily  be  obtained  and  have  been  studied  in  greater 
detail.  In  the  following  pages  we  shall  confine  our  attention  to 
the  composition  of  birds7  eggs,  which  is  best  understood.  The  egg 
proper  is  here  surrounded  by  the  so-called  white  of  egg,  which  in 
turn  is  enclosed  in  a  double  membrane,  and  is  covered  by  the  shell. 
These  additional  structures,  however,  are  not  formed  in  the  ovary, 
but  are  produced  during  the  passage  of  the  egg  through  the  oviduct 
from  material,  which  is  -here  secreted  by  the  lining  cells. 

The  Shell. — The  shell  consists  essentially  of  an  organic  matrix  of 
the  character  of  keratin,  which  is  largely  impregnated  with  lime 
salts.  Of  these,  calcium  carbonate  is  the  most  abundant,  and  con- 
stitutes about  90  per  cent,  of  the  weight  of  the  entire  shell.  In 
addition,  we  find  a  small  amount  of  magnesium  carbonate,  as  also 
phosphates  of  both  elements.  Water  is  present  to  the  extent  of 
only  about  1  per  cent.  The  pigments  met  with  in  birds'  eggs 
are  closely  related  to  the  biliary  pigments,  and,  like  these,  are 
derived  from  the  common  pigment  of  blood.  The  oorhodc'ln,  which 
presents  a  reddish  or  brownish-red  color,  is  supposedly  identical 
with  haematoporphyrin  ;  while  the  blue  or  green  pigment,  which  is 
termed  oocyanin,  is  composed  partly  of  biliverdin,  and  is  in  part  a 
blue  derivative  of  bilirubin. 

The  membranes  of  birds'  eggs  consist  essentially  of  keratin,  but 
contain  also  a  small  amount  of  mineral  salts,  of  which  calcium 
phosphate  is  the  most  abundant. 

In  fishes  and  amphibia  the  egg  envelope  is  represented  by  a  trans- 
parent gelatinous  material,  which  seems  to  consist  almost  exclusively 
of  mucin.  In  the  invertebrates  chitin  and  skeletins  take  the  place 
of  the  keratin  of  birds'  egg*,  but  in  some  the  latter  also  is  found. 

The  weight  of  the  shell  and  membranes  in  the  case  of  hens'  eggs 
represents  about  9  to  11  per  cent,  of  the  total  weight  of  the  egg, 
while  the  albumen  constitutes  about  60.5  per  cent,  and  the  yolk, 
viz.,  the  ovum  proper,  the  remaining  29  per  cent.  The  total  weight 
of  hens'  eggs  may  vary  between  40  and  70  grammes. 

The  Albumen. — The  albumen  or  white  of  egg,  as  obtained  directly 
from  the  raw  egg,  appeai-s  as  a  faintly  yellow,  exceedingly  viscid, 
semi  liquid  material.  On  microscopical  examination  this  can  be 
shown  to  consist  of  compartments,  which  are  limited  by  very 
delicate  membranes,  and  enclose  the  albumen  proper.  These 
membranes  are  continuous  with  the  so-called  chalazse  and  the 
membranes  immediately  beneath  the  shell,  and  are,  like  these, 
composed  of  keratin. 

The  albumen  proper  may  be  separated  from  its  membranous  con- 
stituents by  pressing  the  material  through  a  cloth,  and  then  appears 
:i-  an  opalescenl  fluid,  which  is  only  slightly  viscid,  and  can  be 
filtered  without  much  difficulty.  It-  reaction  is  distinctly  alkaline, 
and  the  specific  gravity  about    1.045.      On   boiling,  it  coagulates  to  a 

compad  ma--,  which  in   the  case  of  liens' eggs   is  entirely  opaque. 

In   some    birds,  however,  such    a-    the    SWalloW,  the   crow,    the    finch, 


424  THE  GLANDULAR   ORGANS. 

etc. — i.  e.,  in  true  nesting  birds — the  albumen  remains  transparent, 
owing  to  the  formation  of  alkaline  albuminates.  Such  albumen  has 
been  termed  tata-albumen.  It  may  be  produced  artificially  by  placing 
hens'  eggs  in  a  10  per  cent,  solution  of  sodium  hydrate  for  two  or 
three  days,  when  a  gradual  diffusion  of  alkali  occurs  into  the  albu- 
men. On  subsequent  boiling,  this  appears  like  true  tata-albumen. 
Analysis  of  the  albumen  of  hens'  eggs  has  given  the  following 

results  : 

Per  cent. 

Water 80.00-86.68 

Solids 13.32-20.00 

Albumins 11.50-12.27 

Extractives 0.38-  0.77 

Glucose 0.10-  0.50 

Fats  and  soaps traces 

Mineral  salts 0.30-  0.66 

Lecithins  and  cholesterin traces 

According  to  Poleck  and  Weber,  the  mineral  ash  has  the  follow- 
ing composition,  calculated  for  100  parts: 

Sodium  (Na.,0) 23.56-32.93 

Potassium  (K.,0) 27.66-28.45 

Calcium  (CaO) 1.74-  2.90 

Magnesium  (MgO) 1.60-  3.17 

Iron  (Fe203) 0.44-  0.55 

Chlorine  (CI) 23.84-28.56 

Phosphoric  acid  (P205) 3.16-4.83 

Carbonic  acid  (C02) 9.67-11.60 

Sulphuric  acid  (S()3)      1.32-  2.63 

Silicic  acid  (Si02) 0  28-0.49 

Fluorine  (Fl) traces 

Of  these  constituents,  the  large  amount  of  sodium  chloride  is  espe- 
cially noteworthy,  and  shows  in  itself  that  the  albumen  is  in  reality 
a  secretory  product,  and  does  not  represent  a  mere  transudation  from 
the  blood-plasma. 

One  portion  of  the  bases  is  in  combination  with  the  albumins  of 
the  albumen,  while  the  remainder  exists  in  the  form  of  sulphates, 
phosphates,  and  notably  carbonates. 

The  slightly  yellow  color  of  albumen  is  referable  to  the  presence 
of  a  lipochrome,  which  can  be  demonstrated  on  spectroscopic  exam- 
ination. 

The  Albumins. — The  albumins  of  albumen  are  largely  represented 
by  the  so-called  ovalbumins  ;  in  addition,  globulins  are  found,  as 
also  a  mucoid,  which  is  known  as  ovomucoid. 

Ovalbumins. — According  to  Gautier  and  others,  the  albumen  con- 
tains three  ovalbumins,  which  are  termed  a-,  /?-,  and  ^-ovalbumin, 
respectively.  They  are  all  closely  related  to  the  common  serum- 
albumin  of  the  blood-plasma,  but  differ  from  this  in  several  impor- 
tant particulars.  When  introduced  into  the  circulation  as  such, 
they  are  eliminated  in  the  urine  as  foreign  matter.  They  are  pre- 
cipitated if  a  sufficient  amount  of  hydrochloric  acid  is  added,  but 
are  soluble  in  an  excess  with  much  greater  difficulty  than  serum- 


THE  REPRODUCTIVE  GLANDS.  425 

albumin.  Alcohol  and  ether  rapidly  destroy  their  solubility.  The 
difference  in  their  degree  of*  rotation  on  polariscopic  examination, 
as  compared  with  serum-albumin,  is  seen  below  : 

Serum-albumin a  ( I>)  f>2.6°-64.6° 

Ovalbumin-a a  (D)  33.1° 

Ovalbumin-/? all))  53. 6° 

Ovalbumin-? a  (D)  70.8° 

The  coagulation-point  of  the  three  ovalbumins  collectively,  in  a 
0.3  per  cent,  solution,  is  at  56°  C.  That  of  the  individual  sub- 
stances is  given  as  72°  C,  76°  C,  and  82°  C,  respectively.  The 
elementary  composition  of  the  different  forms  is  probably  much  the 
same,  and  is  represented  by  the  following  figures  (Hammarsten) :  C, 
52.25;  H,  6.90;  N,  15.25;  S,  1.67-1.93;  O,  23.67-23.93  percent. 

Hofmeister  obtained  the  albumins  in  crystalline  form  by  slow 
evaporation  of  their  solution  in  a  dilute  solution  of  ammonium  sul- 
phate. On  fractional  crystallization  the  different  forms  may  then 
be  obtained.  The  crystals  contained  0.55  per  cent,  of  calcium  phos- 
phate, which  is  apparently  present  in  molecular  combination. 

Isolation". — To  isolate  the  ovalbumins  conjointly,  the  albumen, 
after  separation  from  its  membranes,  is  diluted  with  two  and  one-half 
times  its  volume  of  water.  A  slight  turbidity  thus  results,  which  is 
filtered  off.  The  liquid  is  saturated  with  magnesium  sulphate  in 
substance  at  a  temperature  of  20°  C,  which  causes  precipitation 
of  the  globulins.  After  filtration  sodium  sulphate  is  further  added 
to  saturation,  at  the  same  temperature.  The  ovalbumins  separate 
out  on  standing.  They  are  filtered  off,  dissolved  in  water,  and  freed 
from  salts  by  dialysis.  On  evaporation  in  a  vacuum,  at  a  tempera- 
ture of  from  40°  to  50°  C,  they  are  obtained  in  pure  form. 

To  isolate  tin;  albumins  in  crystalline  form,  the  albumen  is 
beaten  to  a  froth  and  allowed  to  drip.  The  drippings  are  treated 
with  an  equal  volume  of  a  saturated  solution  of  ammonium  sul- 
phate, and  freed  from  globulins  by  filtration.  The  filtrate  is  then 
placed  in  a  shallow  vessel  and  is  allowed  to  evaporate  at  the  tem- 
perature of  the  room.  The  material,  which  thus  separates  out  is 
dissolved  in  water,  treated  with  a  saturated  solution  of  ammonium 
sulphate  until  the  solution  becomes  turbid,  and  is  allowed  to  stand. 
The    resulting  crystals  can  be  purified  by  a  repetition  of  the  process. 

Globulins. — The  globulins  of  the  albumen  represent  only  about 
7  per  cent,  of*  the  total  amount  of  albumin.  Different  forms  appar- 
ently exist,  of  which  one  is  said  to  coagulate  at  47°  C.  and  another 

at  <J7°  C.      They  may  be  isolated  as  described   above. 

Ovomucoid. — The  mucoid  substance  which  can  be  isolated  from 
the  albumen  of  hens'  eggs  is  pre-enf  in  considerable  amount,  consti- 
tuting about    10  per  cent,  of  the  total  solids.      According  to  Morner, 

it  cntain-  12.65  percent,  of  nitrogen  and  2.2  per  cent,  of  sulphur. 
On  boiling  with  dilute  mineral  acide  it    yields  a  reducing  substance, 

which  may  be  of  the  character  of  glucosaniin,  and  is  derived  from  a 

chondroitin-sulphuric  acid  radicle. 


426  THE  GLANDULAR   ORGANS. 

The  substance  cannot  be  precipitated  with  the  common  mineral 
acids,  acetic  acid,  and  potassium  ferrocyanide,  nor  by  salting  with 
sodium  chloride,  magnesium  sulphate,  or  sodium  sulphate.  Tan- 
nic acid,  phosphotungstic  acicl,  ammoniacal  subacetate  of  lead 
solution,  alcohol,  and  ammonium  sulphate,  when  added  to  satura- 
tion, cause  the  substance  to  separate  out.  It  is  soluble  in  water, 
and  is  not  coagulated  by  boiling.  On  evaporating  its  solutions  to  dry- 
ness it  is  rendered  insoluble  in  cold  water,  but  dissolves  on  boiling. 

Isolation. — To  isolate  the  ovomucoid,  the  albumen  is  diluted 
with  water,  as  above,  slightly  acidified  with  acetic  acid,  and  boiled. 
The  coagulable  albumins  are  thus  coagulated  and  filtered  off.  The 
filtrate,  which  still  gives  the  biuret  reaction,  owing  to  the  presence 
of  the  mucoid,  is  concentrated  and  precipitated  with  alcohol,  or 
saturated  with  ammonium  sulphate.  The  mucoid  is  filtered  off  and 
can  then  be  purified  by  repeated  solution  in  water  and  reprecipita- 
tion  with  alcohol. 

The  Yolk. — The  yolk  of  the  egg  represents  the  ovum  proper.  It 
is  surrounded  by  a  delicate  membrane — the  membrana  pellucida — 
which  supposedly  consists  of  keratin  or  a  closely  related  substance. 
Owing  to  the  extensive  development  of  the  protoplasmic  portion  of 
the  cell  proper,  the  germinal  vesicle  is  found  at  the  extreme  periph- 
ery of  the  yolk,  immediately  beneath  the  limiting  membrane.  It 
occupies  the  centre  of  the  discus  proligerus  or  cicatricula,  which 
rests  upon  a  flask-like  cavity  with  a  long,  narrow  neck  that  extends 
to  the  centre  of  the  yolk,  and  is  occupied  by  the  so-called  white  yolk. 
This  surrounds  the  cicatricula  and  also  forms  a  layer  along  the  pe- 
riphery of  the  yolk,  immediately  beneath  the  vitelline  membrane. 
It  contains  albumins,  nucleins,  lecithins,  potassium  salts,  and  possibly 
also  traces  of  glycogen,  though  this  is  doubtful. 

When  broken,  the  yolk  constitutes  a  creamy,  viscid  material,  of 
an  orange-yellow  color,  which  forms  an  emulsion  with  water,  and  is 
coagulated  by  alcohol  and  on  boiling.  Its  reaction  is  feebly  alka- 
line. On  microscopical  examination  it  is  seen  to  consist  of  innumer- 
able spherules,  some  of  which  are  rich  in  fats  and  lipochromes, 
while  others,  which  are  smaller,  are  colorless,  transparent,  semi- 
crystalline  structures  of  an  albuminous  character.  In  the  eggs  of 
certain  amphibia  and  fishes  distinctly  crystalline  bodies  are  further 
met  with,  which  are  spoken  of  as  yolk  platelets,  and  are  analogous 
to  the  aleuron  granules  of  seeds.  As  has  already  been  mentioned,, 
they  probably  consist  of  a  compound  of  albumins  with  lecithins  and 
nucleins.  The  ichthidin,  which  is  found  in  carp  eggs,  and  which  in 
amorphous  form  is  known  as  ichthulin,  belongs  to  this  category.1 

1  From  recent  researches  of  Levene  it  appears  that  different  forms  of  ichthulin 
exist.  The  ichthulin  of  carp  eggs  thus  yields  a  reducing  substance  on  hydrolytic 
decomposition,  while  that  of  the  cod  apparently  contains  no  carbohydrate  radicle. 
The  latter,  on  treating  with  alkalies,  yields  a  paranucleinic  acid,  which  is  similar 
to  vitellinic  acid  (see  below).  Elementary  analysis  of  the  two  forms  has  given  the 
following  results:  Ichthulin  of  carp  eggs  (Walter):  C,  53.52;  H,  7.6;  N,  15.63; 
S,  0.41 ;  P,  0.43  ;  Fe,  0.10  ;  O,  22.19  per  cent.  Ichthulin  of  codfish  eggs  :  C,  52.44  ; 
H,  7.45  ;  N,  15.96  ;  S,  0.92  ;  P,  0.65  ;  Fe  and  O,  22.58  per  cent. 


THE  REPRODUCTIVE  GLANDS.  427 

The  same  holds  good  of  the  ichthin  of  shark  eggs  and  the  emydin 
of  tortoise  eggs. 

As  I  have  stated,  the  yolk  of  hens'  eggs  represents  about  ->f)  per 
cent,  of  the  entire  weight.  Its  actual  weight  may  thus  vary  between 
8.7  and  20.3  grammes.  The  general  composition  of  the  yolk  is  seen 
in  the  following  analyses,  which  are  taken  from  Gautier  : 

Per  cent. 

Water 47.19-51.49 

Solids 48.51-42.81 

Fats  (olein,  palmitin,  and  stearin) 21.30-22.84 

Vitellin  and  other  albumins 15.63-15.76 

Lecithins 8.43-10.72 

Cholesterin 0.44-  1.75 

Cerebrin 0.30 

Mineral  salts 3.33-  0.36 

Coloring-matter  \ 0  553 

(t  hi  cose  I 

Analysis  of  the  mineral  salts,  calculated  for  100  parts  of  ash,  has 
given  the  following  results  (Poleck  and  Weber)  : 

Sodium  (Na20) 5.12-  6.57 

Potassium  (K20) 8.05-  8.93 

Calcium  (CaO) 12.21-13.28 

Magnesium  (MgO)      2.07-  2.11 

Iron  (FeA)      1.19-  1.45 

Phosphoric  acid,  free  (P205) 5.72 

Phosphoric  acid,  combined 63.81-66.70 

Silicic  acid  (Si02) 0.55-  1.40 

Chlorine traces 

Of  the  mineral  constituents,  the  large  amount  of  calcium  and 
phosphoric  acid  is  especially  noteworthy.  Soluble  phosphates, 
however,  are  not  found  as  such  in  the  yolk.  The  amount  of  potas- 
sium and  sodium,  it  will  be  observed,  is  much  smaller  than  in  the 
albumen. 

The  Albumins. — Our  knowledge  of  the  individual  albumins  which 
arc  found  in  the  yolk  is  still  very  imperfect.  But  it  appears  from 
recent  researches  that  they  are  represented  notably  by  nucleo-albu- 
mins,  which  in  turn  may  be  combined  with  lecithins  to  form  com- 
plex lecithalbumins.  This,  however,  is  not  proved,  and  it  is  as- 
sumed by  some  that  the  lecithins  which  are  obtained  so  commonly 
together  with  the  albumins  do  not  exist  in  chemical  combination, 
but  arc  to  l>c  regarded  as  contaminations.  The  best  known  repre- 
sentative of  the  nucleo-albumins  of  the  yolk  is  the  so-called  ovo- 
vitellin.    True  nucleins  do  not  occur  in  the  yolk. 

Ovovitellin. —  Formerly  this  was  regarded  as  a  globulin,  but  it  is 
now  known  to  be  a   nudco-albumin  in  which  an  albuminous    radicle 

\-  combined  with  a  paranuclein — /'.  e.,a  nuclein  which  does  not  yield 

DUcleinic  bases  on  decomposition  with  mineral  acids.  The  substance 
ha-  thu-  far  not  been  obtained  free  from  lecithins,  and  it  is  for  this 
reason  that  the  latter  i-  thought  by  some  to  be  present  ill  chemical 
combimit  ion. 


428  THE  GLANDULAR   ORGANS. 

Of  the  character  of  the  albuminous  radicle  which  is  present 
in  combination  with  the  paranuclein  nothing  is  known.  The 
paranuclein  has  recently  been  studied  in  detail  by  Levene  and 
Alsberg.  They  term  it  avivitellinic  acid,  and  give  the  following 
figures  to  express  its  elementary  composition  :  C,  32.31 ;  H,  5.58  ; 
N,  13.13;  P,  9.88;  S,  0.3266;  O,  38.28  per  cent,  In  addition 
they  found  0.57  per  cent,  of  iron,  which  is  present  in  organic  com- 
bination. This  is  especially  interesting  in  view  of  the  fact  that 
Bunge  also  obtained  a  nuclein  from  the  yolk  of  hens'  eggs,  which 
contained  iron,  and  which  he  termed  hcematogen,  as  the  product  must 
of  necessity  be  concerned  in  the  formation  of  the  blood-coloring  mat- 
ter of  the  developing  animal.  The  elementary  composition  of  Bunge's 
hsematogen,  however,  is  different  from  Levene's  avivitellinic  acid, 
viz.,  C,  42.11  ;  H,  6.08;  N,  14.73;  S,  0.55;  P,  5.19;  Fe,  0.29 ; 
and  O,  31.05  per  cent,  Its  relation  to  ovovitellin  is  at  present 
not  clear,  but  it  is  manifestly  closely  related  to  avivitellinic  acid. 

The  albuminous  radicle  of  avivitellinic  acid  manifestly  contains 
the  protamin  group,  as  Levene  was  able  to  isolate  both  arginin  and 
histidin  from  its  decomposition-products,  which  resulted  on  boiling 
the  substance  for  seventy-two  hours  with  a  20  per  cent,  solution  of 
hydrochloric  acid.  Whether  or  not  lysin  is  also  present  remains  to 
be  seen.  The  amount  of  arginin  and  histidin  obtained  was  so  small, 
however,  that  it  is  scarcely  warrantable  to  assume  that  a  protamin 
constitutes  the  entire  albuminous  radicle,  as  in  the  case  of  the  nii- 
cleins  which  can  be  obtained  from  certain  fishes.  The  substance 
gives  Millon's  reaction,  moreover,  which  is  not  obtained  with  pro- 
tamins.  The  biuret  reaction  was  positive.  For  a  more  detailed 
account  of  Levene's  most  interesting  work,  and  a  description  of  the 
method  which  was  employed  for  isolating  the  avivitellinic  acid,  I 
must  refer  the  reader  to  his  article.1 

The  ovivitellin  as  it  is  obtained  from  the  yolk  contains  about  25 
per  cent,  of  lecithin.  It  is  soluble  in  dilute  solutions  of  the  neutral 
salts,  and  in  very  dilute  (1  pro  mille)  solutions  of  hydrochloric  acid, 
and  the  hydrates  and  the  carbonates  of  the  alkalies.  In  water  it 
is  insoluble,  and  accordingly  is  precipitated  from  its  solutions  on 
copious  dilution.  On  prolonged  contact  with  water  its  properties 
are  changed,  and  it  is  converted  gradually  into  an  albuminate-like 
substance.  Sodium  chloride  when  added  to  saturation  causes  only 
a  partial  precipitation.  When  slowly  heated  in  its  solutions  of 
neutral  salts  it  coagulates  between  70°  and  75°  C. ;  when  rapidly 
heated,  coagulation  is  retarded  until  80°  C  is  reached.  On  diges- 
tion with  gastric  juice  ovivitellin  yields  a  paranuclein — avivitellinic 
acid.  From  the  ovivitellin  of  the  eggs  of  the  bony  fishes  a  gluco- 
paranuclein  may  be  obtained. 

Elementary  analysis  of  the  ichthulin  of  carp  eggs  has  given  the 
following  results  :  C,  53.52  ;  H,  7.6  ;  N,  15.63  ;  O,  22.19  ;  S,  0.41  ; 
P,  0.43  ;  and  Fe,  0.1  per  cent.     For  the  ichthulin  of  codfish  eggs 

1  Zeit.  f.  physiol.  Cliem.,  vol.  xxxi.  pp.  543-556. 


THE  REPRODUCTIVE  GLANDS.  429 

Levene  found  C,  52.44  ;  H,  7.45  ;  N,  15.96  ;  S,  0.92 ;  P,  0.65  j  Fe 
and  O,  22.58  per  cent.  On  treating  with  alkalies  a  substance  is 
obtained  from  this  latter  form,  which  is  quite  similar  in  compo- 
sition to  avivitellinic  acid,  as  is  seen  from  the  figures:  C,  32.56; 
II,  6;  X,  14.03;  S,  0.146;  P,  10.34  per  cent.  It  is  termed  ich- 
thulinic  aeid  (see  also  page  428). 

Isolation. — To  isolate  the  ovivitelliu  from  the  yolk,  it  is  well  to 
employ  a  large  number  of  eggs.  The  yolks  are  thoroughly  mixed 
with  an  equal  volume  of  a  10  per  cent,  solution  of  sodium  chloride, 
and  arc  completely  extracted  with  ether,  by  shaking,  viz.,  until  no 
more  coloring-matter  can  be  removed,  and  the  sodium  chloride 
solution  has  become  perfectly  transparent.  This  is  then  diluted 
with  twenty  times  its  volume  of  water,  and  the  ovovitellin  thus 
precipitated.  To  purify  the  substance  further,  it  is  dissolved  re- 
peatedly in  a  10  per  cent,  saline  solution,  and  reprecipitated  with 
water.  It  is  washed  finally  with  alcohol  and  ether,  and  dried  over 
sulphuric  acid. 

The  Fats. — The  fat  of  the  yolk  consists  almost  entirely  of  olein, 
palmitin,  and  stearin.  As  a  whole,  it  contains  a  somewhat  smaller 
amount  of'  carbon  than  ordinary  fat,  which  may  be  due  to  the  pres- 
ence of  mono-  and  diglycerides,  or  to  the  presence  of  a  fatty  acid 
which  contain-  less  carbon  than  usual.  On  saponification  Lieber- 
mann  obtained  40  per  cent,  of  oleic  acid,  38.04  per  cent,  of  palmitic 
acid,  and  15.21  per  cent,  of  stearic  acid. 

The  lipochromes  or  luteins  of  the  yolk  can  be  isolated  as  follows  : 
the  fats  of  the  yolk  are  saponified  by  boiling  with  an  alcoholic 
solution  of  sodium  hydrate.  The  alcohol  is  then  evaporated.  The 
remaining  solution  is  treated  with  calcium  chloride,  which  trans- 
forms the  soluble  sodium  salts  into  the  corresponding  insoluble  cal- 
cium salts.  On  cooling,  the  soaps  are  extracted  with  petroleum- 
ether,  which  takes  up  the  lipochromes.  On  evaporation  they  are 
then  obtained  in  pure  form.  The  entire  process  of  isolation  must 
be  carried  on  in  the  absence  of  daylight,  as  otherwise  the  pigments 
are  decomposed  after  being  separated  from  the  fats.  In  birds'  eggs 
a  yellow  lipochrome,  vitettolutein,  is  notably  found,  but,  in  addi- 
tion, traces  of  a  red  pigment  of  the  same  order,  which  is  termed 
vitellorubin,  may  also  be  encountered.  This  latter  cannot  well  be 
obtained  by  extracting  the  soaps  with  petroleum-ether  directly,  but 
it  i-  necessary  previously  to  decompose  these  with  a  mineral  acid. 

Lecithins. — The  general  properties  of  the  lecithins  have  been  con- 
sidered in  a  previous  chapter  (page  65). 

Isolation. — To  isolate  the  lecithins,  the  method  of  Ziilzer  may 

be  conveniently  employed.  To  this  end,  the  yolks  of  a  large  num- 
ber of  eggs  (fifty  or  more)  arc  first  extracted  with  ether  by  shaking, 
until  the  ethereal  solution  takes  up  no  more  pigment.     The  ethereal 

extracts  are  united,  the  ether  is  distilled  off,  and  the  oil  tillered  oil" 
at  the  temperature  of  the  body.  This  is  best  accomplished  in  a 
thermostat       The   yellow,  somewhat   frothy  material  which   remains 


430  THE  GLANDULAR   ORGANS. 

on  the  filter  is  dissolved  in  as  little  ether  as  possible,  and  precipi- 
tated with  acetone.  The  precipitate  is  collected  on  a  filter  and 
washed  with  acetone  until  the  wash-acetone  dissolves  no  more 
cholesterin.  The  residue  is  again  dissolved  in  a  small  amount  of 
ether  or  benzol.  To  this  solution  an  excess  of  absolute  alcohol  is 
added,  when  on  standing  a  white  amorphous  substance  separates 
out,  which  can  be  obtained  in  crystalline  form  by  solution  in  hot 
alcohol  and  cooling ;  this  apparently  consists  of  tripalmitin.  After 
filtration  the  pure  lecithin  can  then  be  obtained  from  the  ether- 
alcoholic  solution  by  precipitating  with  acetone,  as  before,  or  by  dis- 
tilling off  the  alcohol  and  ether.  The  resulting  material  is  dried  in 
the  vacuum:  Its  phosphorus  varies  between  3.7  and  4.1  per  cent, 
in  amount. 

Incubation. — Of  the  chemical  changes  which  take  place  during 
the  process  of  fertilization,  and  in  which  the  nucleus  of  the  ovum  is 
primarily  concerned,  we  know  nothing.  But  there  can  be  no  doubt 
that,  in  fishes  at  least,  the  protamin  radicle  of  the  nucleins  of  the 
spermatozoa  plays  an  important  part.  As  a  result,  the  reproductive 
function  of  the  ovum,  which  previously  has  remained  dormant, 
now  manifests  itself  in  the  mysterious  morphological  changes  which 
the  cell  undergoes,  and  which  end  in  the  production  of  an  organism 
that  is  morphologically  and  chemically  like  its  parents. 

In  mammals  the  food-stuffs  which  are  required  by  the  devel- 
oping organism  are  constantly  supplied  through  the  blood  of  the 
mother-animal,  but  in  the  lower  forms  of  life  they  are  furnished 
directly  in  the  egg  itself.  These  products  have  been  studied  in 
some  detail  in  the  foregoing  pages,  and  we  have  seen  that  they  are 
in  part,  at  least,  specific  of  the  egg,  and  do  not  occur  elsewhere  in 
the  animal  body.  This  holds  good  more  especially  of  the  albu- 
mins, and  it  follows  that  all  those  forms  that  enter  into  the  com- 
position of  the  various  tissues  must  of  necessity  be-produced  from 
the  pre-existing  forms  during  the  development  of  the  young  animal. 
The  fats  may,  in  part,  be  utilized  directly  in  the  construction  of  the 
fats  of  the  embryo,  but  to  a  large  extent,  no  doubt,  they  represent 
the  principal  form  of  energy  which  is  placed  at  the  disposal  of  the 
developing  organism.  Carbohydrates,  as  such,  are  practically  lack- 
ing among  the  food-stuffs  of  the  egg,  and  must  hence  be  formed 
synthetically.  That  glycogen  can  be  demonstrated  in  the  tissues 
of  the  embryo  at  a  very  early  date,  has  already  been  stated,  which 
proves  in  itself  that  the  animal  organism  is  not  dependent  upon 
the  ingested  carbohydrates  for  its  glycogen  supply.  As  nuclear 
nucleins,  moreover,  do  not  occur  in  the  egg,  it  follows  that  these 
also  must  be  formed  from  other  albuminous  substances,  and  there 
can  be  little  doubt  that  the  paranucleins  are  here  of  prime  impor- 
tance.  The  salts  which  are  required  by  the  developing  organism 
are,  as  has  been  seen,  present  in  the  egg  in  abundance. 

The  essential  factor,  however,  which  is  necessary  to  development 


THE  REPRODUCTIVE  GLAXDS.  4.J1 

after  fertilization,  is  an  abundant  supply  of  oxygen,  and  a  tempera- 
ture of  about  40°  C.  The  requisite  amount  of  oxygen  is  obtained 
from  the  air  by  a  process  of  diffusion  through  the  shell.  In  return 
carbon  dioxide  is  eliminated,  together  with  a  small  amount  of 
nitrogen.  These  respiratory  changes  are  but  slight  in  the  begin- 
ning, but  gradually  increase.  Water  also  is  given  off,  and,  as  a 
result,  the  weight  of  the  ego-  diminishes.  The  increase  of  the  solids 
in  the  developing  animal  is,  of  course,  accompanied  by  a  correspond- 
ing diminution  of  those  of  the  egg  itself. 

Systematic  chemical  examinations  of  the  ovum  in  its  various 
stages  of  development  have  thus  far  not  been  made.  Liebermann 
appears  to  be  the  only  one,  indeed,  who  has  attempted  the  problem. 
His  principal  results  may  be  summarized  as  follows  :  during  the 
first  stage  of  development  tissues  are  formed,  which  are  very  rich  in 
water  ;  later,  however,  the  amount  of  water  decreases.  The  abso- 
lute amount  of  substances  which  are  soluble  in  water  steadily  in- 
creases, while  their  relative  amount  diminishes  as  compared  with 
the  remaining  solids.  After  the  fourteenth  day  a  large  increase  in 
the  amount  of  fat  is  noted,  while  previously  this  remains  fairly  con- 
stant.  The  amount  of  soluble  albumins  and  albuminoids  increases 
steadily  and  in  such  a  manner  that  the  absolute  quantity  increases, 
while  their  relative  amount  remains  nearly  constant.  Up  to  the 
tenth  day  no  collagen  is  found,  but  after  the  fourteenth  day  a  sub- 
stance i-  present  which  on  boiling  with  water  yields  a  material  sim- 
ilar to  cartilaginous  glutin.  A  mucinous  substance  is  found  about 
the  sixth  day,  but  it  subsequently  disappears.  The  amount  of 
haemoglobin  steadily  increases  in  its  relation  to  the  body-weight. 

The  chemical  composition  of  the  allantoic  fluid  and  the  amniotic 
fluid  has  already  been  considered  (page  344). 

The  placenta  has  not  as  yet  been  studied  in  detail,  but  it  is  likely 
that  its  greater  portion  consists  of  collagen,  in  accordance  with  its 
fibrous  structnre.  In  its  marginal  zone  two  pigments  have  been 
encountered  which  apparently  are  related  closely  to  bilirubin  and 
biliverdin,  and  are  derivatives  of  haemoglobin.  The  orange  pigment 
may  be  obtained  in  crystalline  form,  while  the  green  pigment,  which 
has  been  termed  fuematochlorine,  is  amorphous. 


CHAPTER   XXII. 

THE  DUCTLESS  GLANDS. 

THE  THYROID  GLAND. 

Of  the  function  of  the  thyroid  gland  very  little  is  known.  Its- 
removal  leads  sooner  or  later  to  the  death  of  the  animal.  This 
is  preceeded  by  various  symptoms  of  nerve  irritation,  and  in  man 
by  the  development  of  marked  anaemia,  impairment  of  the  mental 
powers,  general  prostration,  and  curious  trophic  disturbances  of 
the  skin,  which  are  associated  with  an  increased  development  of 
the  subcutaneous  connective  tissue  and  a  coincident  increase  in 
the  amount  of  mucin.  As  a  result  the  skin  appears  swollen  and 
oedematous,  constituting  the  condition  known  as  myxoedema.  Sim- 
ilar results  occur  if  from  any  cause  the  gland  atrophies.  If,  how- 
ever, the  resection  of  the  gland  is  partial,  deleterious  results  do 
not  necessarily  follow.  It  is  thus  manifest  that  the  function  of  the 
organ  is  a  most  important  one,  and  it  is  interesting  to  note  that  the 
apparent  antitoxic  properties  of  the  gland  persist  even  after  its 
removal  from  the  body  and  subsequent  desiccation.  The  various 
symptoms  which  have  just  been  described  as  following  extirpation 
of  the  organ  may  thus  be  prevented  by  administration  of  the 
dried  gland,  and  in  cases  of  atrophy  a  curative  effect  may  similarly 
be  obtained.  It  is  noteworthy,  moreover,  that  the  administration 
of  the  substance  has  a  marked  effect  upon  the  nitrogenous  metabo- 
lism of  the  body,  which  is  distinctly  increased,  and,  if  continued, 
emaciation  results  although  an  abundant  amount  of  food  is  ingested, 
and  digestion  and  resorption  remain  unimpaired.  In  some  instances 
true  diabetes  develops.  In  addition,  an  increased  pulse-rate  and 
heightened  blood-pressure  are  commonly  observed,  and  even  sud- 
den death  may  occur  during  the  use  of  the  substance. 

That  these  curious  properties  of  the  thyroid  gland  should  also 
find  expression  in  its  chemical  composition  suggests  itself  at  once. 
Numerous  attempts  have  accordingly  been  made  to  isolate  the 
"active  principle"  of  the  organ,  and  to  a  certain  extent  these  at- 
tempts have  been  successful.  Baumann  and  Roos  thus  succeeded 
in  isolating  from  the  gland  a  substance  which  has  manifestly  the 
same  properties  as  the  entire  organ  in  preventing  the  deleterious 
results  which  follow  its  extirpation  or  atrophy,  and  which  also  has 
the  same  effect  upon  the  circulation  and  the  nitrogenous  metabolism. 
This  substance  Baumann  termed  thyroiodine,  from  the  fact  that  it 
contains  iodine  in  organic  combination.    It  is  obtained  by  boiling  the 

432 


THE  THYROID   GLASD.  433 

gland  for  several  hours  with  a  10  per  cent,  solution  of  sulphuric 
acid,  and  by  subsequently  extracting  the  insoluble  residue  with  90 
per  cent,  alcohol.  It  is  insoluble  in  water  and  acids,  but  readily 
dissolves' in  dilute  alkaline  solutions,  from  which  it  is  precipitated 
by  adding  an  excess  of  an  acid.  The  substance,  as  first  obtained  by 
Baumann,  contained  9.3  per  cent,  of  iodine  and  a  small  amount  of 
phosphorus.  This  latter,  however,  he  regarded  as  a  contamination, 
and  he  expressed  the  opinion  that  future  researches  would  show  that 
the  chemically  pure  body  contained  even  more  iodine  than  the  crude 
product  he  obtained.  Regarding  the  chemical  nature  of  the  thv- 
roiodine,  which  itself  does  not  give  the  biuret  reaction,  Baumann 
supposed  that  it  existed  in  the  gland  in  combination  with  an  albu- 
min, viz..  as  a  thvro-iodoglobulin-  or  albumin.  This  has  since 
been  proved,  through  the  researches  of  Oswald,  who  succeeded  in 
extracting  from  the  gland  a  globulin  which  contains  the  entire 
amount  of  iodine,  and  which  yields  Baumann's  thyroiodine  on 
decomposition  with  mineral  acids.  This  substance  is  termed  thy- 
reoglobulin. 

Thyreoglobulin  is  found  in  the  colloid  material  of  the  organ 
together  with  a  nucleo-albumin,  which  latter,  however,  is  present 
in"  much  smaller  amounts.  Its  quantity  is  directly  dependent 
upon  the  amount  of  the  colloid,  and  is  thus  subject  to  variation.  In 
the  human  gland  it  normally  represents  about  one-third  of  the 
weight  of  the  dried  organ,  viz.,  1.6  grammes.  In  that  of  sheep_  it 
has  been  found  in  the  proportion  of  1  : 2,  or  2  : 3,  as  compared  with 
the  total  amount  of  solids,  and  similar  results  have  been  obtained 
in  the  pig.  Its  general  elementary  composition  in  animals  of  the 
-;iin(  species  is  quite  constant,  and  varies  but  little  indeed  in 
animals  of  different  species.  The  amount  of  iodine,  however, 
which  i-  present  in  organic  combination  is  subject  to  fairly  wide 
variations.  This  is  shown  in  the  following  analyses,  which  are  taken 
from  Oswald: 

ox.  n  Man,  Man, 

Pig.  Sheep.  Ox.  normal.      colloid  goitre. 

C      52.21  52.32  52.45  51.85  52.02 

II      6.83  7.02  6.93  6.88  6.91 

N      16.59  15.90  15.92  15.49  15.32 

I       0.46  0.39  0.86  0.34  0.07 

S      1.86  1.95  1.83  1.87  1.93 

O      22.15  22.42  22.01  23.57  23.75 

it  i-  thus  seen  that  in  colloid  goitres  especially  small  amounts  of 
iodine  are  apparently  present.  This,  however,  is  only  relatively 
tin-  case,  and  in  accordance  with  the  presence  of  a  larger  quantity 
of  colloid  the  total  amount  of  the  thyreoglobulin  is  increased, 
and  we  find  thai  the  absolute  amount  of  iodine  is  actually  larger 
than  normal.  It-  quantity  can  artificially  be  increased  by  the 
ingestion  of  iodine  or  iodide-  ;i-  such,  so  that  it  is  apparent  that 
the  globulin  i-  capable  of  binding  a  certain  amount  that  is  intro- 
duced from   without. 


434  THE  DUCTLESS  GLANDS. 

The  iodine,  however,  is  manifestly  not  a  constant  component  of 
the  globulin,  and  may  be  absent  altogether.  This  is  especially 
interesting  in  view  of  the  fact  that,  whereas  the  iodized  globulin 
possesses  all  the  specific  properties  of  the  entire  gland,  the  non- 
iodized  substance  is  inert.  An  adequate  explanation  of  this  curious 
phenomenon  cannot  at  present  be  given,  but  we  may  imagine  that 
in  those  instances  in  which  the  iodine  is  normally  absent  its  place 
may  be  taken  by  some  other  halogen,  or  a  compound  halogen  which 
has  escaped  observation.  To  conclude  that  the  iodized  substance 
is  not  the  active  principle  of  the  gland,  on  the  basis  that  the 
glands  of  some  animals  contain  no  iodine,  and  that  its  amount  is 
more  or  less  variable  and  can  artificially  be  increased,  is  scarcely 
warrantable.  It  is  conceivable  that  in  sucklings,  for  example,  in 
which  iodine  is  commonly  absent,  a  compound  halogen  takes  its 
place,  and  is,  for  the  time  being  at  least,  fully  capable  of  preventing 
the  development  of  the  complex  of  symptoms  which  we  term  ca- 
chexia strumipriva.  Further  researches,  however,  are  necessary  to 
explain  satisfactorily  the  apparent  contradiction.  The  fact  that  the 
thyreoglobulin  of  those  glands  in  which  it  is  especially  abundant 
contains  a  relatively  smaller  amount  of  iodine  than  others  in  which 
less  globulin  is  present,  can  readily  be  explained  on  the  basis  that 
the  total  amount  of  iodine  is  here  distributed  over  a  larger  quantity 
of  the  globulin,  and,  as  has  been  stated,  the  absolute  amount  is  here 
larger  than  in  normal  glands. 

In  its  general  properties  thyreoglobulin  resembles  the  common 
globulin  of  the  blood.  It  differs,  from  this,  however,  in  the  fact 
that  it  is  precipitated  from  its  saline  solutions  on  the  addition  of 
dilute  acids.  In  this  respect  it  resembles  the  myosin  of  muscle- 
tissue.  Its  point  of  coagulation,  in  a  10  per  cent,  solution  of  mag- 
nesium sulphate,  varies  between  65°  and  67°  C.  It  is  precipitated 
from  its  solutions  by  the  addition  of  an  equal  volume  of  a  saturated 
solution  of  ammonium  sulphate. 

On  decomposition  with  dilute  acids  it  yields  the  thyroiodine  of 
Baumann,  but  contains  more  iodine,  viz.,  14.29  per  cent.,  and,  like 
its  mother-substance,  it  is  free  from  phosphorus. 

Thyreo-nucleo-albumin. — The  nucleo-albumin  which,  as  I  have 
stated,  is  found  in  association  with  thyreoglobulin  in  the  colloid 
material  of  the  gland,  but  is  present  in  much  smaller  amounts, 
contains  0.16  per  cent,  of  phosphorus.  In  a  10  per  cent,  solu- 
tion of  magnesium  sulphate  it  coagulates  at  73°  C.  On  digestion 
with  gastric  juice  a  nuclein  is  split  off,  which  contains  xanthin- 
bases.  The  substance  is  free  from  iodine,  and  is  physiologically 
inert.  It  is  precipitated  from  its  solutions  by  salting  with  am- 
monium sulphate  to   saturation. 

For  a  further  description  of  the  two  albumins,  and  of  the 
methods  which  may  be  employed  for  their  isolation,  I  must  refer 
the  reader  to  Oswald's  paper.1 

xZeit.  f.  pliysiol.  Chem.,  vol.  xxvii.  p.  14. 


THE  ADRENAL   GLANDS.  435 

The  extractives  of  the  thyroid  gland  are  represented  by  traces 
of  xanthin,  hypoxantbin,  lenein,  succinic  acid,  and  paralactic  acid. 
In  addition,  notable  quantities  of  kreatinin  may  be  obtained. 

THE  ADRENAL  GLANDS. 

<  >f  the  function  of  the  adrenal  glands,  nothing  definite  is  known- 
Their  integrity,  however,  is  essential  to  life,  and,  as  in  the  case  of  the 
thyroid,  their  removal  leads  to  the  death  of  the  animal.  It  has  been 
noted,  moreover,  that  the  injection  of  blood  from  a  dog  which  has 
died  as  a  result  of  the  operation,  into  the  circulation  of  a  second 
animal  that  has  been  operated  in  the  same  manner,  will  hasten  the 
fatal  end,  while  in  normal  dogs  no  deleterious  results  are  observed. 
It  has  hence  been  concluded  that  the  glands  normally  furnish  a 
secretion  which  renders  certain  metabolic  products  innocuous,  and 
that  the  fatal  result  which  follows  the  removal  of  the  organs  is  the 
result  of  an  auto-intoxication. 

In  man,  disease  of  the  adrenal  glands  leads  to  the  complex  of 
Bymptoms  which  is  commonly  known  as  Addison's  disease,  and  like- 
wise  results  in  death  ;  but  as  in  the  case  of  the  thyroid  gland,  it  has 
been  observed  that  the  fatal  issue. may  here  also  at  least  be  retarded 
by  the  administration  of  an  aqueous  extract  of  the  organs.  Experi- 
ment- with  such  extracts  have  further  shown  that  the  gland  contains 
a  substance  which  lias  a  very  marked  effect  upon  the  blood-pressure, 
raising  this  far  beyond  the  normal.  This  substance  is  found  in  the 
medullary  portion  of  the  glands.  Further  investigations  have  then 
demonstrated  the  existence  of  a  chromogen  which  on  exposure  to  the 
air  in  aqueous  solution  yields  a  beautiful  carmin-colored  pigment, 
which,  like  its  mother-substance,  is  soluble  in  water.  The  same 
result  is  reached  at  once  on  treating  with  chlorine-,  bromine-,  or 
iodine-water.  This  chromogen  is  present  in  the  intracellular  fluid  of 
the  medullary  portion  of  the  gland.  When  this  is  extracted  with  a 
dilute  acid,  a  violet-red  precipitate  results  on  the  addition  of  an 
-  of  ammonia,  which  suggests  that  the  pigment  is  of  a  basic 
nature. 

\\  ith  a  solution  of  ferric  chloride  the  juice  that  can  be  expressed 
from  the  glands  gives  a  bright  emerald-green  color.  This  reaction 
has  b.cn  referred  to  the  supposed  presence  of  pyrocatechin, but  thus 
for  this  has  never  I n  isolated. 

Modern  researches  lead  to  the  conclusion  that  the  blood-pressure- 
raising  constituent  of  the  gland,  as  also  the  chromogen,  which  gives 
i'i-e  to  the  carmin  color  ami  the  pyrocatechin  reaction,  are  identical 
bodies.  Abel,  who  claims  to  have  isolated  the  blood-pressure-raising 
principle  of  the  glands,  states  that  this  must  in  all   probability  be 

classed  with  the  pyrrol  compounds  or  with  the  pyridin  bases  or  alka- 
loid-. II. •  \\:1-  unable,  however,  to  obtain  the  -ub<tance,  which  he 
term-    epmephrin,  in    pure    form.      IVrociitchin    could    not    be  split 

off  on  boiling  with  an  acid,  but  he  states  thai  a  carmin-red  pigment 


436  THE  DUCTLESS  GLANDS. 

can  be  separated  from  the  sulphate  of  the  active  principle  without 
destroying  its  power  to  raise  the  blood-pressure. 

v.  Furth,  on  the  other  hand,  who  likewise  attempted  to  isolate 
the  blood-pressure-raising  constituent  of  the  gland,  states  that  Abel's 
epinephrin  is  merely  an  inactive  foreign  substance,  contaminated 
with  the  active  principle  which  he  claims  to  have  isolated,  and 
which  he  terms  suprareniyi.  v.  Furth,  however,  was  likewise  not 
able  to  obtain  his  suprarenin  in  pure  form. 

Of  late,  Takamine  also  has  announced  that  he  has  succeeded  in 
isolating  the  blood-pressure-raising  constituent  of  the  gland  in  a 
stable  and  crystalline  form.  This  substance  he  terms  adrenalin. 
From  a  preliminary  report,  which  he  has  kindly  placed  at  my  dis- 
posal, I  abstract  the  following  : 

Adrenalin  is  a  light,  white  crystalline  substance,  of  a  slightly 
bitter  taste,  leaving  a  numb  feeling  on  the  tongue  where  it  has  been 
applied.  When  dry,  it  is  perfectly  stable.  On  heating,  it  turns 
brown  at  205°  C.  At  207°  C.  it  melts,  and  is  at  the  same  time 
decomposed.  Its  reaction  is  slightly  alkaline.  In  cold  water  it  is 
soluble  with  difficulty,  but  more  readily  so  in  hot  water.  From  its 
hot  solution  it  crystallizes  out  on  cooling.  It  is  easily  soluble  in 
acids  and  alkalies,  but  not  in  ammonia  or  solutions  of  the  alkaline 
carbonates.  Upon  the  addition  of  ferric  chloride  its  solutions  are 
colored  a  fine  emerald-green,  which  changes  to  a  purple  and  then  to 
a  carmin  red  upon  the  careful  addition  of  caustic  alkali.  Strong  acids 
prevent  the  reaction,  limiting  the  change  of  color  to  a  dirty  yellow- 
ish-green. It  reduces  silver  salts  and  auric  chloride  very  ener- 
getically, while  the  liquid  at  the  same  time  turns  red.  This  also 
occurs  on  treating  with  oxidizing  agents,  such  as  potassium  ferri- 
cyanide  and  potassium  bichromate.  The  usual  alkaloidal  reagents 
do  not  precipitate  the  substance.  With  acids  it  forms  salts,  but 
these  have  not  been  obtained  in  crystalline  form. 

The  elementary  composition  of  the  substance  has  not  been  ascer- 
tained. 

The  blood-pressure-raising  power  of  adrenalin  is  very  marked. 
The  amount  pro  kilo  of  body-weight  which  is  required  to  raise  the 
blood-pressure  14  Hgmm.  beyond  the  normal  is  one-millionth  part 
of  a  gramme,  and  distinct  physiological  effects  can  be  obtained  by  the 
administration  of  even  one-fourteenth  millionth  part  of  a  gramme. 

In  addition  to  the  blood-pressure-raising  constituent,  the  adrenal 
glands  contain  collagen,  which  enters  into  the  composition  of  the 
supporting  tissue ;  albumins,  which  have  not  as  yet  been  studied  in 
detail ;  and,  further,  a  substance  which  apparently  is  related  closely 
to  jecorin,  and  yields  fatty  acids,  neurin,  glycerin-phosphoric  acid, 
and  glucose  on  hydrolytic  decomposition  with  baryta-water.  Be- 
sides, we  meet  with  lecithins  and  a  small  amount  of  inosit.  Benzoic 
acid,  hippuric  acid,  and  biliary  acids  are  not  present,  as  was  formerly 
supposed. 


ofthe Tyolkof  em?  "f  ''""'  chloride  ■©lotion  j  ':>,  bfVwlutionoMuteln  (etheieal  extract 

437 


INDEX. 


ACCIPENSERIN,  70 
Acetic  acid,  93 

in  stomach  contents,  116 
tests  for,  123 
Acetone,  94,  267 

estimation  of,  268 

tests  for,  267 
Achromatin,  304 
Achroodextrin,  61,  111,  166 
Acid,  acetic,  93 

adenylic,  75 

arachinic,  415 

avivitellinic,  428 

barbituric,  82 

benzoic,  259 

bilianic,  152 

bilirnbinic,  157 

biliverdinic,  158 

bromo-phenyl-mercapturic,  88 

butyric,  9.J,  116 

capronic,  93 

carbamic,  87 

carnic,  175,  351 

chenocholalic,  153 

chenotaurocliolic,  150 

chi tonic,  50 

cholalic,  151 

cliolanic,  152,  153 

choleic,  153 

choleo-camphoric,  152 

cholesteric,  152 

choloidinic,  1  ■">— 

cholonic,  1  19 

chondroitin-eulphuric,  45,   47,  256, 
348 

citric,    117 

damalic,  263 
damaliiric,  263 
dehydrocholalic,  152 
dehydrocholeic,  153 
deaoxycholalic,  L52 
diacetic,  94,  266 
dialui 

dioxy-phenyl-aoetic,  260 
eleidinic,  95 
ethylene-succinic,  72 
fatty,  93,  264 
fellic,  163 

ferri-albuminic,  (02 
formti 
galactonic,  55.  57 


Acid,  gluconic,  55,  256 

glucuronic,  55,  91,  255,  321,  384 
glutaminic,  37,  86,  191 
glutaric,  95 

glycerin-phosphoric,  65,  298 
glycocholic,  89,  148 
glycolic,  94 
glycoluric,  82 
glycosuric,  262 
guanidin-butyric,  72 
guano-biliary,  149 
guanylic,  75,  405 
haematinic,  158,  332 
liippuric,  87,  97,  257 
honiogentisinic,  261 
hydantoic,  82,  85 
hydantoin-hydroparacumaric,  88 
hydrocinnamic,  97 
hydroparacu marie,  95,  201,  260 
hydurilic,  82 
hyocholalic,  153 
hyoglycocbolic,  149 
hyotaurocholic,  150 
icbthulinic,  429 
inosinic,  75,  367 
isobilianic,  152 
kynurenic,  263 
lactic,  94,  115,  356,359 
lfflvulinic,  56,  323 
laurinic,  415 
leucinic,  94 
lithofellic,  153 
lithuric,  363 
lysuric,  74 
mannonic,  5 1 
manno-saccharinic,  55 
methyl-hydantoinic,  88 
tnucinic,  55,  57 
myristinic,  168,  415 
nuclei 1 1 i<-,  .'54,  74 
oleic.  64 

ornithuric,  74,  87,  260 
oxalic,  95,  241 
oxaluric,  82,  241 
oxy-amygdalic,  260 
oxy-hydroparacumaric,  95 
oxy-protonic,  88 
oxy-proto-Hulphonic,  '-'>x 
palmitic.  64,  98 
parabanic,  ho,  82 
paralactic,  269 

130 


440 


INDEX. 


Acid,  para-oxy-benzoic,  96 

para-oxy-pbenyl-acetic,  95,  201,  260 

para-oxy-phenyl-glycolie,  260 

para-oxy-phenyl-lactic,  95,  260 

para-oxy-phenyl-propionic,  95,  260 

peroxy-pro tonic,  38 

phenaceturic,  97,  259 

phenyl-acetic,  97,  201 

phenyl-propionic,  97,  201 

phosphor-car nic,  351 

phyllocyanic,  222 

plasminic,  76,  77 

propionic,  93 

pyrocatecliuic,  262 

pyrocliolesteric,  152 

saccliarinic,  55,  91,  256 

saccharo-lactonic,  55 

sarcylic,  75 

skatol-carbonic,  207,  254 

sperma-nucleinic,  75 

stearic,  93 

tartronic,  83 

taurocarbaminic,  88,  271 

taurocholic,  88,  149 

thyminic,  76,  77,  323 

thymo-nucleinic,  75,  323 

trioxy-phenyl-propionic,  260 

tyrosin-hydantoinic,  88 

uramic,  88 

uramido-benzoic,  88 

uric,  80,  81,  233 

nrocaninic,  263 

urochloralic,  255 

uroleucinic,  262 

uroxanthinic,  262 

urrhodinic,  262 

valerianic,  93 

xanthylic,  75 

yeast-nncleinic,  75 
Acids,  biliary,  145 
Acrolein,  65 

Adamkiewicz'  reaction,  34 
Addison's  disease,  435 
Adenin,  78,  240,  366 

isolation  of,  363 

tests  for,  366 
Adenylic  acid,  75 
Adipocere,  391 
Adipose  tissue,  3S9 

analysis  of,  390 
origin  of,  391 
significance  of,  392 
Adrenal  glands,  435 
Adrenalin,  Takamine's,  436 
Albumen  of  birds'  eggs,  423 

albumins  of,  424 

analysis  of,  424 

ovalbumins  of,  424 

ovomucoid  of,  425 

tata,  424 
Albumin  of  Bence  Jones,  289 
Albuminates,  49 

alkaline,  49 


Albuminates  of  iron,  401 
Albuminoids,  46 

digestion  of,  179 
Albumins,  30,  41 

behavior   toward  neutral  salts  and 
alcohol,  31 
toward  polarized  light,  33 

classification  of,  40 

coagulated,  49 

coagulation  of,  32 

color-reactions  of,  34 

crystallization  of,  30 

decomposition  of,  37 

denaturization  of,  33 

derived,  48 

digestion  of,  168 

elementary  composition  of,  30 

estimation  of,  in  milk,  415 
in  plasma,  316 
in  urine,  290 

molecular  size  of,  38 

native,  41 

nitrogenous  derivatives  of,  69 

of  the  albumen,  424 

of  the  blood,  309 

of  the  lymph,  342 

of  the  milk,  411 

of  the  muscle-tissue,  347 

of  the  nerve-tissue,  370 

of  the  urine,  284 

of  the  yolk,  427 

precipitation  of,  36 

solubility  of,  31 

structural  composition  of,  39 

synthesis  of,  26,  38 

tests  for,  in  the  urine,  286 
Albuminuria,  284 

physiological,  285 
Albumoid,  378,  385 

isolation  of,  385 
Albumose-plasma,  313 
Albumoses,  49,  169 

analysis  of,  183 

in  the  urine,  285 

primary,  50,  169 

secondary,  50,  169 

reactions  of,  186,  187 

tests  for,  in  the  urine,  288 
Aldehydase,  106,  402 
Aldoses,  54 
Aleuron  crystals,  30,  42 

granules,  30,  42 
Alkapton,  262 
Allantoic  acid,  82 
Allantoin.  82,  83 

in  the  urine,  243 

isolation  of,  244 
Allanturic  acid,  82 
Allituric  acid,  82 
Alloxan,  80,  82,  83 
Alloxanic  acid,  82 
Alloxantin,  82 
Alloxuric  bases,  43,  75,  77 


INDEX. 


441 


Amido-acids,  85 

Amido-nitrogen,  37 

Amido-thio-lactic  acid,  88 

Aniidulin,  111,  166 

Ammonia,  estimation  of,  in  the  urine,  232 

in  the  blood,  322 
Ammonium  purpurate,  82,  83 
Amniotic  fluid,  analysis  of,  344 
Amphikreatin,  363 
Amphikreatinin,  84 
Amphopeptone,  51,  170 
Amygdalin,  26 
Amyiodextrin,  60 
Amyloid,  48 
Amylolytic  ferment   of  pancreatic  juice, 

133 
Amylopsin,  138 
Amylum,  60 
Animal  cell,  301 

fat,  389 

gum,  44 

sinistrin,  44 
Anti-albumid,  170,  176 
Anti-albumoses,  50 
Antipeptone,  51,  175 

preparation  of,  195 
Aqueous  humor,  analysis  of,  343,  377 
Arabinose,  62,  284 
Arachinic  acid,  415 
Arbacin,  71 
Arbutin,  25 
Arginin,  37,  69,  72,  84 

in  the  spleen,  406 
Arnold's  test  for  diacetic  acid,  266 
Aromatic  constituents  of  the  urine,  247 

oxy-aci<ls,  95 

in  the  urine,  260 
Arterin,  327 

Ascherson's  baptogenic  membrane,  408 
Asparagin,  190 
Asparaginic  acid,  37,  86,  190 
isolation  of,  191 

BACTERIAL  action  in  the  intestinal 
tract,  196 
decomposition    of    biliary    constitu- 
ents, 202 
of  fats  202 
Barbituric  acid,  82 
Bayer's  indigo-purpurin,  251 
Bence  Jones  albumin,  289 
Benzoic  acid,  isolation  of,  from  hippuric 

a<-id,  25fl 

Bezaar  stones,  L53 

Bile,  !  11 

acids  of  (see  also  Biliary  adds),  I  15 
amount  of,  143 
chemical  composition  of,  144 
crystallized,  of  Plainer,  147 
general  properties  of,  143 
mucinous  body  of,  1 t"> 
pigments  of,  155,  156 

in  the  urine,  297 


Bile,  secretion  of,  142 

significance  of,  142 
Biliauic  acid,  152 
Biliary  acids,  145,  202,  297 
formation  of,  145 
isolation  of,  147 
tests  for,  147 
constituents,  bacterial  decomposition 

of,  202 
iron,  164 
Bilicyanin,  161 
Bilifuscin,  158,  161 
Bilihumin,  162 
Biliprasin,  158,  161 
Bilipurpurin.  161 
Bilirubin,  22,  156  _ 
isolation  of,  159 
tests  for,  158 
Bilirubinic  acid,  157 
Biliverdin,  158,  160 
isolation  of,  160 
Biliverdinic  acid,  158 
Bilixanthin,  161 
Biuret  reaction,  34 
Blood,  306 

amount  of,  309 

chemical  composition  of,  310 

examination  of,  309 
coagulation  of,  324 
color  of,  307 
extractives  of,  322 
fat  in,  322 

fibrin-ferment  in,  319 
fibrinogen  in,  313 
glucose  in,  321 
glycogen  in,  321 
leucocytes  of,  322 
odor  of,  308 

physical  characteristics  of,  307 
pigments  of,  328 
plaques  of,  324 
reaction  of,  309 
red  corpuscles  of,  327 
serum-albumin  of,  316 
serum-globulin  of,  314 
specific  gravity  of,  308 
taste  of,  308 
urea  in,  322 
Blood-pigments  in  the  urine,  294 
Blood-plasma,  307,  312 
Blood-serum,  307,  317 
Boas'    method  of  estimating  lactic  acid, 
123 
test,  120 

for  lactic  acid,  122 
Bone,  386  _ 

analysis  of,  387 
Bone-marrow,  388 
Boucher's  crystals,  420 
Brain-tissue,  analysis  of,  370 
Bromo-phenyl  mercapturic  acid,  88 
Brown-Sequard's  elixir,  421 
Brucke's  method  of  isolating  pepsin,  128 


442 


INDEX. 


Brunner's  glands,  secretion  of,  139 
Bufidin,  398 
Butter,  415 
Buttermilk,  411 
Butyric  acid,  93 

in  stomach  contents,   116 

tests  for,  123 

nACHEXIA  strumipriva,  434 
\J     Cadaverin,  74 

in  the  urine,  299 
Caffein,  78,  240 
Calcium-thrombosin,  320 
Cane-sugar,  58 
Capronic  acid,  93 
Carbamic  acid,  87 
Carbohsemoglobin,  335 
Carbohydrates,  53 

fermentation  of,  56 

of  the  urine,  275 

synthesis  of,  in  plants,  24 
Carbon  dioxide  haemoglobin,  335 
methsemoglobin,  337 

monoxide  haemoglobin,  335 
Carnic  acid,  351 

in  antipeptone,  175 
Carniferrin,  351 
Carnin,  78,  240 

in  muscle-tissue,  366 

isolation  of,  363 

properties  of,  367 

relation  of,  to  hypoxanthin,  366 

tests  for,  367 
Cartilage,  383 

albumoid  in,  385 

analysis  of,  384 

chondroitin-sulphuric  acid  in,  384 

chondromucoid  in,  385 

embryonic,  383 

mineral  salts  in,  386 
Casein,  411 

digestion  of,  178 

estimation  of,  415 

isolation  of,  414 

origin  of,  414 

properties  of,  411 
Caseoses,  50 
Castoreum,  398 
Cavazzani's  method  to  remove  albumins 

from  the  blood,  317 
Cell -globulins,  324 
Celluloses,  60,  61 
Cement-substance  of  teeth,  388 
Cerebrin,  57,  373 

isolation  of,  374 

properties  of,  374 
Cerebrosides  in  nerve-tissue,  372 
Cerebrospinal  fluid,  analysis  of,  344 
Cerumen,  398 
Cetin,  64 
Cetylid,  373 

Cliarcot-Leyden  crystals,  420 
Cheese,  412 


Chenocholalic  acid,  153 

Chenotaurocholic  acid,  150 

Chitin,  46,  56,  389 

Chitonic  acid,  56 

Chitose,  56 

Chlorides,  estimation  of,  in  the  urine,  219' 

Chlorine-hunger,  217 

Chlorophane,  381 

Chlorophyl,  19,  21 

chemical  nature  of,  21 

granules,  21 

hydride,  24 
Cholagogues,  143 
Cholalic  acid,  151 
Cholanic  acid,  152,  153 
Cholecyanin,  161 
Choieglobins,  164 
Choleic  acid,  153 
Choleo-camphoric  acid,  152 
Cholesteric  acid,  152 
Cholesterilins,  162 
Cholesterin,  67 

in  nerve-tissue,  376 

in  the  bile,  162 

in  the  urine,  298 

isolation  of,  163 

tests  for,  163 
Choletelin,  161,  291 
Cholin,  65 

Choloidinic  acid,  152 
Cholonic  acid,  149 
Chondrin,  47,  385 
Chondroitin,  46,  384_ 
Chondroitin-sulphuric  acid,  45,  47 
in  cartilage,  384 
isolation  of,  385 
properties  of,  384 
Chondromucoid,  385 

isolation  of,  385 
Chond rosin,  45,  46,  384 
Choroid,  381 
Chromatin,  304 

Chromogen  of  adrenal  glands,  435 
Chromophanes,  381 
Chyle,  analysis  of,  343 
Chymosin,  130,  139 

estimation  of,  132 

isolation  of,  131 

properties  of,  130 

tests  for,  131 
Chymosinogen,  130 

estimation  of,  132 

test  for,  131 
Citric  acid  in  milk,  417 
Clabber,  416 
Clupein,  69 

Coagulated  albumins,  49 
Coagulation  of  the  blood,  324 
rapidity  of,  326 
theories  of,  324 
Collagen,  47 
Collagen-sugar,  155 
Colloid,  45 


INDEX. 


443 


Colloidal  platinum,  20 
Colostrum,  417 

Compound  glycocolls  in  the  urine,  256 
Conchiolin,  47,  48 
Conjugate  glucuronates,  96 
in  the  urine,  255 
sulphates,  96 

estimation  of,  220 
in  the  urine,  248 
Connective  tissue,  embryonic,  382 
Copper  test  for  uric  acid,  237 
Cornea,  377 
Corpora  lutea,  422 
Cream,  411 
a-Crotonic  acid,  95 
Crusokreatinin,  84,  363 
Crusta  phlogistica  (seu  inflammatoria), 

327 
Crystalline  lens,  378 

albumins  of,  378 
albumoid  of,  378 
analysis  of,  378 
crystallins  of,  378 
Crystallins,  378 
Cyan-methaemoglobin,  337 
Cyanurin,  251 
Cyclopterin,  71 
Cystei'n,  88 

in  the  urine,  272 
Cystin,  88 

in  the  kidneys,  406 
in  the  liver,  404 
in  the  urine,  273 
isolation  of,  274 
properties  of,  274 
Cystinuria  associated   with    diaminuria, 

299 
Cytoglobin,  305 
(  Vtosin,  75,  323 

DAM  A  LIC  acid,  263 
Damaluric  acid,  263 
Dehydrocholalic  acid,  152 
Dehydrocholeic  acid,  183 
Denaturization,  169 
Denniges'  (est  for  uric  acid,  237 
Dentin 
I  terived  albumins,  48 

met'a  membrane,  377 
I  '•      xycliolalic  acid,   162 

Deutero-albumoaes,  60,  169,  171,  186 

l)extrins,  UK,  6] 

in  tin-  arine, 
Dezi 

Diabetes,  277 
Diacetic  -Kid.  '.»  1 

in  the  arine,  266 
for,  246 

I >i;ilnrit   acid,  82 
Diamino-nitrogen,  88 

I »i:imiiis  in  tin-  urine,  299 

isolation  ol 
DiamiBuria,  27:; 


Dibutyl-diethylene  diamin,  38 

Diethylene  diamin,  38 

Differential  density  method  of  estimating 

sugar,  280 
Digestion,  165 

of  albumins,  168 

of  albuminoids,  179 

of  carbohydrates,  165 

of  fats,  180 

ofproteids,   178 
Digestive  fluids,  108 

glands,  405 
Dioxv-phenyl-acetic   acid  in  the  urine, 

260 
Disaccharides,  57 
Diureids,  81 
Dry  pancreas,  138 
Dulcite,  54 
Dun  lop's   method   of   estimating  oxalic 

acid,  243 
Dysalbumose,  50 
Dyslysin,  152 

EAR,  the,  381 
Egg-albumin,  41 
Eggs,  422 

albumen  of,  423 

albumins  of,  424 

fats  of,  429 

incubation  of,  430 

lecithins  of,  42y 

lipochromes  of,  429 

ovomucoid  in,  425 

ovovitellin  in,  427 

shell  of,  423 

yolk  of,  426 
Ehrlich's  reaction,  268 
Elastin,  47 
Elastoidin,  47,  48 
Elastoses,  50 
Eleidin  granules,  394 
Eleidinic  acid,  95 
Emulsin,  105 
Emydin,  427 
Enamel  of  teeth,  388 
Encephalin,  375 
Enteric  juice,  140 
Enzymes.     Sec  Ferment*. 
Eosinophilic  crystalloids,  30 
Epiguanin,  78 
Epinephrin,  Abel's,  435 
Episaroin,  78,  240 
Erythrodextrin,  60,  111,  166 
Esbach's  reagent,  129,  290 
Ethylene-succinic  acid,  72 
Ethylenimin,  120 

Ethyl  Sulphide  in  the  urine,  271 

Excretin  in  the  feces,  208 
Extractives  "f  nerve  tissue,  376 
of  the  blood,  822 

Of  the  liver,    Mil 

of  the  lymph,  848 

of  the  milk,  417 


444 


INDEX. 


Extractives  of  the  thyroid  gland,  435 
Eye,  377 

FAT,  63 
analysis  of,  390 

animal,  389 

bacterial  decomposition  of,  202 

chemistry  of,  64 

digestion  of,  180 

estimation  of,  in  the  milk,  416 

melting-point  of,  390 

of  birds'  egg,  429 

of  the  blood,  322 

of  the  liver,  404 

of  the  lymph,  341 

of  the  milk,  415 

of  the  muscle-tissue,  368 

of  the  urine,  298 

origin  of,  63,  391 
in  milk,  415 

significance  of,  393 

synthesis  of,  in  plants,  26 
Fatty  acids,  93 

estimation  of,  in  the  urine,  264 
isolation  of,  from  the  urine,  264 

degeneration,  391,  404 

infiltration,  404 

tissue  (see  also  Adipose  tissue),  389 
Feces,  204 

amount  of,  204 

bacteria  in,  205 

chemical  composition  of,  205 

color  of,  204 

consistence  of,  204 

crystals  in,  205 

excretin  in,  208 

form  of,  204 

hydrobilirubin  in,  207 

macroscopical  constituents  of,  205 

microscopical  constituents  of,  205 

odor  of,  204 

reaction  of,  205 

serolin  in,  208 

stercobilin  in,  207 

stercorin  in,  208 
Fehling's  test  for  sugar,  278 
Fellic  acid,  153 
Fermentation,  acetic,  198 

alcoholic,  198 

butvric,  198 

lactic,  198 

method  of  estimating  sugar,  281 

test  for  sugar,  279 
Ferments,  20,  98 

amylolytic,  105 

chemical  composition,  103 

classification,  104 

coagulating,  105 

general  properties  of,  101 
reactions  of,  103 

inverting,  105 

mode  of  action,  104 

of  the  liver,  402 


Ferments  of  the  lymph,  343 

of  the  muscle-tissue,  352 

of  the  pancreatic  juice,  136 

of  the  urine,  298 

oxidizing,  106 

proteolytic,  105 

reversible  action  of,  21,  103 

steatolytic,  105 

tissue-,  106 
Ferratin,  402 
Ferri-albuminic  acid,  402 
Fertilization  of  eggs,  430 
Fibrin,  48,  319 

estimation  of,  320 

formation  of,  319 

isolation  of,  320 

properties  of,  320 

test  for,  in  the  urine,  290 
Fibrin-ferment,  319,  324 

chemical  nature  of,  319,  324 

isolation  of,  319 

properties  of,  319 
Fibrin  of  Henle,  419 
Fibrinogen,  41 

isolation  of,  from  the  blood,  313 

properties  of,  314 

tissue-,  305 
Fibrinoglobulin,  41,  324 
Fibrinoplastic  substance  of  Schmidt,  314 
Fibroin,  47,  48 
Fibrous  tissue,  elastic,  383 
reticulated,  383 
white,  382 
yellow,  383 
Fish-scales,  388 
Fleischl's  ruemometer,  334 
Floridins,  329,  338  _ 
Fluorides  in  the  urine,  219 
Folin's  method  of  estimating  urea,  231 

uric  acid,  238 
Food-stuffs  of  plants,  23 
Formic  acid,  93 

Freund's  method  of  estimating  the  acid- 
ity of  the  urine,  215 
Fructose.     See  Lcevulose. 
Fuscin,  381,  395 

pADUShiston,  71 
\J     Galactonic  acid,  55,  57 
Galactose,  54,  57,  373 
Gallois'  test  for  inosit,  360 
Gases  of  the  blood,  312 

of  the  intestinal  contents,  201 
of  the  lymph,  343 
of  the  muscle-tissue,  367 
of  the  stomach  contents,  132 
of  the  urine,  299 
Gastric  digestion,  168 
juice,  114 

acidity  of,  115 

amount  of,  114 

analysis  of,  115 

chemical  composition  of,  115 


IXDEX. 


445 


Gastric  juice,  ferments  of,  124 

gases  of,  132 

hydrochloric  acid  of,   117 
lactic  acid  in,  122 
pro-enzymes  of,  124 
sulphocyanides  in,  132 
(Gelatin,  47 

<  relatoses,  50 

Gerhardt's  test  for  diacetic  acid,   267 

for  urobilin,  293 
Giacosa's  pigment,  251  » 

<  Hands,  adrenal,  435 

digestive,  405 

ductless,  432 

gastric,  405 

intestinal,  405 

lymph,  405 

mammary,  406 

reproductive,  419 

salivary,  405 
<ilobin,  320 

isolation  of,  330 
Globulinoses,  50 
Globulins,  41 

cell-,  324,  327 

secondary,  of  Hammarsten,  315 
Glueite,  54 
Glucocyamidin,  84 
Glucocyamin,  84 
Glucolysis,  321 
Glucolvtic  ferment  of  Lepine,  134,  139, 

321  * 
Gluconic  acid,  55,  256 

<  ilucoproteids,  44 
Glucosamin,  45,  46,  56 

relation  to  glucuronic  acid,  256 
Glucose,  54,  57 

estimation  of,  in  the  urine,  280 

in  the  liver,  404 

in  the  muscle-tissue,  356 

in  the  urine,  276 

tests  for,  278 
Glucosides  in  nerve-tissue,  372 

synthesis  of,  in  plants,  25 

<  rlucosuria,  276 

<  • I  neuron,  256 
Glucuronates,  conjugate,  91,  255 

<  rlncaronic  acid,  55,  91 

in  the  blood,  321 

in  the  urine,  255 

origin  of,  255,  '.',- 1 

properties  of,  256 

teste  for,  256 
'  Hutamin,  19] 
Glutaminic  add,  37,  86,  191 

isolation  of,  191 
Glntaric  acid,  95 
Glntin,  17 
GIu  tin-peptone,  17!t 
Glycerin-phosphoric  acid,  65 

in  the  urine,  298 
icholic  acid,  88,  1  1- 
Glycocoll,  87,  80,  86,  155,  192 


Glycocoll,  isolation  of,  155 

relation  to  hippuric  acid,  192,  259 

test  for,  192 
Glycogen,  61,  353,  403 

estimation  of,  403 

formation  of,  353 

in  the  blood,  321 

in  the  liver,  403 

in  the  muscle-tissue,  353 

isolation  of,  355 

properties  of,  403 

significance  of,  354 
Glycol ic  acid,  94 
Glycoluric  acid,  82 
Glycosuric  acid,  262 
Gmelin's  test,  158 
Gowers'  luemoglobinometer,  334 
Granulose,  60 
Guanidin,  72,  80,  85 
Guanidin-butyric  acid,  72 
Guanin,  78,  85,  240 

in  muscle-tissue,  365 

isolation  of,  363 

tests  for,  366 
Guano-biliary  acid,  149 
Guanylic  acid,  75,  405 
Gunning's  test  for  acetone,  268 
Gunzburg's    test  for   hydrochloric  acid, 
119 

H.K.MAT1N,  320,  331 
in  the  urine,  295 

isolation  of,  332 
Hsematinic  acid,  158,  332 
Hiematochlorine,  431 
Hsematogen,  400 
Htematoidin,  156,  338 
Hamiatoporphyrin,  22,  330,  337  ■ 

in  the  urine,  295 

preparation  of,  337 
Hfemin,  332 

preparation  of,  332 
Hcemochromogen,  330 

isolation  of,  330 
Ilaemocyanin,  338 
Haemoglobin,  328 

amount  of,  331 

chemical  nature  of,  329,  331 

isolation  of,  331 
ETeemoglobinometer  of  Gowers,  :;.",! 
I  haemoglobins,  46 
Ibemolvinph,  838 
Hsemometer  of  Fleischl,  334 
ETammerschlag's  method  of  determining 

the  specific  gravity  of  blood,  308 
Eaptogenic  membrane  of  Ascherson,  408 
Harnblau,  251 
FTehner-Seeman'a  method  of  estimating 

organic  acids,  124 
ETelicoproteid,   II 
I  teller's  test  for  albumin,  36 

for  blood,  294 

urrhodin,  251 


446 


INDEX. 


Hemi-albumose,  50 
Hemicelluloses,  62 
Hemipeptone,  51,  175 
Henle's  fibrin,  419 
Hepatin,  400 

Hetero-albumoses,  50,  169,  171 
Heteroxanthin,  78,  240 
Hexobioses,  58 
Hexon  bases,  37,  52,  69,  71 

in  antipeptone,  175 
Hexoses,  54,  55 
Hippomelanin,  297,  395 
Hippuric  acid,  87,  97 

estimation  of,  259 

isolation  of,  258 

of  the  urine,  257 

origin  of,  257 

properties  of,  257 

synthesis  of,  258 
Histenzyme,  258 
Histidin,  37,  69,  74 
Histon,  305,  323 

test  for,  in  the  urine,  290 
Histon-plasma,  326 
Hoffmann's  test  for  tyrosin,  189 
Homocerebrin,  374 
Homogentisinic  acid  in  the  urine,  261 

isolation  of,  262 
Hopkin's  method  of  estimating  uric  acid, 

238 
Hoppe-Seyler's  double  pipette,  334 

test  for  xanthin,  365 
Huppert's  test,  159 
Hyalins,  45,  47 
Hyalogens,  44,  45,  389 
Hyalomucoid,  379 
Hyaloplasm,  303 
Hydantoic  acid,  82,  85 
Hydantoin,  82 

Hydantoin-hydroparacumaric  acid,  88 
Hydrations  in  animal  life,  19 
Hydrazons,  55 

Hydrobilirubin  in  the  feces,  207 
Hydrocele  fluid,  analysis  of,  344 
Hydrochloric  acid,  115,  117 

combined,  120 

free,  115,  117 

of  gastric  juice,  115,  117 

quantitative  estimation  of,  120 

secretion  of,  117 

significance  of,  118 

tests  for,  119 
Hydrocinnamic  acid,  97 
Hydrogen  peroxide  in  the  urine,  219 

sulphide  in  the  urine,  299 
Hydroparacumaric  acid,  95 

in  the  intestinal  contents,  201 

in  the  urine,  260 

isolation  of,  207 
Hydroquinon,  25,  96 
in  the  urine,  248 
Hydnrilic  acid,  82 
Hyocholalic  acid,  153 


Hyoglycocholic  acid,  149 
Hyotaurocholic  acid,  150 
Hypobromite  method  of  estimating  urea, 

230 
Hypoxanthin,  78,  240 

in  muscle-tissue,  365 

isolation  of,  363 
Hypoxanthylic  acid,  75 

TCHTHIDIN,  426 
1     Ichthin,  427 
Ichthulin,  44,  427,  428 
Ichthulinic  acid,  429 
llasvay's  reagent,  113 
Incubation  of  eggs.  430 
Indican,  animal,  90 

estimation  of,  253 
in  the  urine,  250 
origin  of,  250 
properties  of,  251 
tests  for,  252 

vegetable,  91 
Indiglucin,  91 
Indigo-blue,  90,  91,  251 
Indigo-purpurin,  251 
Indigo-white,  91 
Indirubin,  251 
Indol,  90,  198 

isolation  of,  206 

tests  for,  199 
Indoxyl,  90 
Indoxyl-red,  251 

Indoxyl  sulphate  (see  also  Indican),  90 
Inosinic  acid,  75 

in  muscle-tissue,  367 
Inosit  in  muscle-tissue,  359 

in  the  urine,  262     . 

isolation  of,  263,  360 

tests  for,  360 
Intestinal  putrefaction,  196 
Inulin,  61 
Invertin,  58 
Invertins,  105 
Invert-sugar,  58 
Iron  albuminate,  401 

in  the  liver,  164 
Isobilianic  acid,  152 
Isocholesterin,  67 
Isomaltose,  58,  59,  61,  111,  166 
Ivory,  388 

TAFFE'S  test  for  indican,  252 
*J      Jecorin  in  nerve-tissue,  376 

KAULOSTEEIN,  67 
Kefir-granules,  58 
Kelling's  test  for  lactic  acid,  122 
Kerasin.     See  Homocerebrin. 
Keratin  in  the  skin,  394 
Keratinoses,  50 
Keratins,  47 
Ketoses,  54 
Kidneys,  406 


INDEX. 


447 


Kjeldahl's  method  of  estimating  nitro- 
gen, 232 
Knapp's  method  of  estimating  sugar,  128 
Knop-Hiifner  method  of  estimating  urea, 

230 
Kornein,  47,  48 
Kossel's  hypothesis,  39 
Kossler- Penny's   method   of    estimating 

phenols,  249 
Kreatin,  84 

in  muscle-tissue,  360 

in  the  urine,  245 

isolation  of,  362 

origin  of,  360 

properties  of,  362 

relation  to  kreatinin,  246 
Kreatinic  leucomains,  84 
Kreatinin,  84 

estimation  of,  246 

in  muscle-tissue,  360 

in  the  urine,  244 

isolation  of,  246 

origin  of,  244 

properties  of,  245,  362 

relation  to  kreatin,  361,  362 

synthesis  of,  246 

tests  for,  246 
Kreatins,  84 

Kiihne's  method  of  isolating  pepsin,  129 
Kvnurenic  acid,  263 
Kynnrin,  263 

LACTALBUMIN,  41,  413 
estimation  of,  415 
isolation  of,  414 
Lactases,  105 
Lactic  acid,  94 

estimation   of,   in   the   stomach 

contents,  123 
in  the  muscle-tissue,  356 
in  tlie  stomach  contents,  115 
isolation  of,  from  muscle-tissue, 

358 
origin  of,  in  muscle-tissue,  357 
properties  of,  359 
significance  of,  356 
tests  for,  122 
Lactoglobulin,  414 
estimation  of,  415 
isolation  of,  414 
Lactose.  58,  59,  416 
estimation  of,  417 
in  the  milk,  418 
in  the  urine,  282 
isolation  of,  282,  116 
Leerulinic  acid,  56,  328 
Leevnlose,  5 1.  57 

in  the  mine,  283 

Laiosi 

Lanolin,  67,  162 
Latham's  hypothesis,  89 
taarinic  acia,  415 
LecUhalbamins,  86 


Lecithins,  65 

digestion  of,  182 

in  bird's'  eggs,  429 

in  muscle-tissue,  368 

in  nerve-tissue,  375 

in  the  urine,  298 

isolation  of,  375,  429 
Legal' s  test  for  acetone,  267 
Leichenwax.     See  Adipocere. 
Leo's  method  of  estimating  hydrochloric 
acid,  121 

sugar.     See  Laiose. 
Lepine's  glucolytic  ferment,  134 
Leucin,  37,  86,  185 

in  the  urine,  270 

isolation  of,  189 

tests  for,  188 
Leucinic  acid,  94 
Leucites,  21 
Leucocytes,  322 

chemical  composition  of,  303,  322 
Leucomains,  91 

kreatinic,  84 

xanthinic,  77 
Leukonuclein,  305 
Leuko-urobilin,  205 
Lichenin,  61 

Lieben's  test  for  acetone,  268 
Liebermann-Burckhard's  test  for  choles- 

terin,  163 
Lignin,  61 

Lilienfeld's  nucleohiston,  322 
v.  Limbeck's  method  of  estimating  the 

alkalinity  of  the  blood,  310 
Lipochrin,  381 
Lipochromes,  65,  338,  429 
Lipochromic  pigments,  338,  429 
Lithofellic  acid,  153 
Lithuric  acid,  263 
Liver,  399 

albuminates  of,  401 

albumins  of,  400 

chemical  composition  of,  401 

extractives  of,  404 

fats  of,  404 

ferments  of,  402 

function  of,  399 

glucose  of,  404 

glycogen  of,  403 

nucleins  of,  400 
Living  matter,   chemical  changes  in,  IS 
(ones  at  work  in,  17 
genera]  composition  of,  17 
Lota-histon,  71 

Lowv's  method  of  estimating  the  alka- 
linity of  the  blood,  310 
Ludwig-Salkowski  method  of  estimating 
uric  acid,  238 
of  estimating  santhin-bases,  241 
Luteins,  the,  of  bird-eggs,  \-'.t 
Lymph,  :::;'.» 

amount  of,  .''.  II 
analyses  of,   343 


448 


INDEX. 


Lymph,  chemical  composition  of,  341 

general  properties  of,  340 
Lymphagogues,  340 
Lymph-glands,  405 
Lysatin,  37,  85 
Ly  satin  in,  37,  85 
Lysin,  37,  69,  73 
Lysuric  acid,  74 

MALONYL-UREA,  82 
Maltase  in  the  pancreatic  juice,  139 
in  the  saliva,  112 
Maltases,  105 
Maltodextrin,  61 
Maltose,  58.  59,  61,  111,  166 

in  the  urine,  283 
Mammary  glands,  406 
Mannides,  synthesis  of,  in  plants,  26 
Mannite,  26,  54 
Mannitides.     See  Mannides. 
Mannonic  acid,  55 
Manno-saccharinic  acid,  55 
Mannose,  54 
Meconium,  208 
Melanins,  395 

in  the  skin,  395 

in  the  urine,  296 
Melanogen,  296 
Membranin,  45,  377 
Messinger-Huppert  method  of  estimating 

acetone,  268 
Metabolism,  29 
Metalbumin,  45 
Methsemoglobin,  336 

sulphide,  337 
Methiemoglobinsemia,  336 
Methyl-glycocoll,  84,  85 
Methyl-guanidin,  85 
Methyl-hydantoin,  82,  85 

in  muscle-tissue,  361 
Methyl-hydantoinic  acid,  88 
Methyl-uracil,  85 
Milk,  407  _ 

albumins  of,  411 

amount  of,  409 

analysis  of,  410 

casein  in,  411 

chemical  composition  of,  410 

citric  acid  in,  417 

coagulation  of,  411 

extractives  of,  417 

fats  in,  415 

general  characteristics  of,  407 

lactalbumin  in,  413 

lactoglobulin  in,  414 

lactose  in,  416 

reaction  of,  410 

skimmed,  411 

specific  gravity  of,  409 
Millon's  reaction,  34 
Mineral  ash,  estimation  of,  in  the  urine, 

219 
Molisch's  test,  35 


Mono-amino  nitrogen,  39 
Monosaccharides,  54 
Mono-ureids,  81,  82 

Morner  and  Sjoqvist's  method  of  estimat- 
ing hydrochloric  acid,  120 
of  estimating  urea,  230 
Mucin,  biliary,  145 

salivary,  110,   112 
Mucinic  acid,  55,  57 
Mucinogen,  110,  112 
Mucinoids,  44,  45" 
Mucins,  44,  45 
Mucoids,  44,  45 

corneal,  379 
Mucous  tissue,  382 
Murexid,  82,  83 

test,  83,  237 
Muse,  398 
Muscarin,  66 

Muscle-albumins,  significance  of,  350 
Muscle-pigments,  353 
Muscle-plasma,  347 
Muscle-serum,  348 
Muscle-stroma,  347,  352 
Muscle-tissue,  346 

albumins  of,  347 

analyses  of,  346 

carnin  in,  366 

fat  in,  367 

ferments  of,  352 

gases  in,  367 

glucose  in,  356 

glycogen  in,  353 

guanin  in,  365 

hypoxanthin  in,  365 

inosinic  acid  in,  367 

inosit  in,  359 

kreatin  in,  360 

kreatinin  in,  360 

lactic  acid  in,  356 

nitrogenous  extractives  of,  360 

nucleins  of,  351 

phosphor-carnic  acid  in,  351 

pigments  of,  353 

plasma  of,  347 

stroma  of,  352 

xanthin  in,  364 

xanthin-bases  in,  363 
Myelin,  66,  372 
Myelin-bodies.  372 
Myo-albumose,  351 
Myogen,  isolation  of,  348 

properties  of,  349 
Myogen-fibrin,  insoluble,  349 

soluble,  349 
Myoglobulin,  351 
Myoproteid,  351 
Myosin,  41,  348,  349 

isolation  of,  349 

properties  of,  349 
Myosin-fibrin,  350 
Myosinogen,  351 
Myosinoses,  50 


ISDEX. 


449 


Myriein,  64 
Myristinic  acid.  163 

in  the  bile,  163 
Myrosin,  10-3 
Myxcedema,  4-">2 

VKoSSIX,  45 

^\      Nerve-tissue,  369 

albumins  in.  370 

cerebri n  in,  373 

cholesterin  in,  376 

encephalin  in,  375 

extractives  of,  376 

general    chemical     composition 

of,  369 
homocerebrin  in,  374 
jecorin  in,  376 
lecithins  in,  375 
myelins  in,  372 
neuridin  in,  376 
neurokeratin  in,  371 
protagon  in,  372 
Neuridin  in  nerve-tissue.  376 
Neu rin,  66 
Neurokeratin,  371 
Neutral  oxygen,  19 

sulphur,  estimation  of,  275 
Nitrates  in  the  saliva,  113 
test  for,  113 
in  the  urine,  219 
test  for,  221 
Nitric  acid  test  lor  albumin,  286 
for  xanthin,  365 
oxide  haemoglobin,  336 
Nitrogen,  estimation  of,  232 
Nitrogenous  equilibrium,  225 
Nuclear  nucleins,  43.  71 
Nucleinic  acids,  43,  74 

bases,  43,  77 
Nuclein  platelets  of  the  Mood,  324 
Nucleins,  11 

digestion  of,  178,  179 
isolation  of,  101 

Of  the  liver.  400 

of  the  muscle-tissue,  351 

Nudeo -albumin-.   13 

test  for,  in  the  urine,  288 

Nucleo-gliicoproteid    of   the    mammary 

gland.  406 

of  the  pancre  is,  105 

Nucli-o-bi-ton,  305 

isolation  of,  322 

properties  of,  323 
Surleons, 

v.  l.uid'T'-  test  for  -  . 

0BERM  IYER'S  test  for  indioan,  262 
Oleic  acid,  61 
Oletn,  61 
Onuphin,  16 

nio,  12.; 
Oornodein,  123 


Ornithin,  72,  88 
Ornithuric  acid.  74.  87,  260 
Osazons,  55 
( )>«  >ns,  55 
Ossein,  3S6 
Ovalbumins,  424 

isolation  of,  425 
Ovaries,  422 
Ovomucoid,  425 

isolation  of,  426 
Ovovitellin,  427 

isolation  of,  429 
properties  of,  428 
Ovum,  422 
( Palate-plasma,  313 
( txalic  acid,  95 

estimation  of,  243 

in  the  urine.  241 

origin  of,  241 
Oxaluric  aeid,  82 

in  the  urine,  241 

origin  of,  241 
Oxidations  in  animal  life,  19 
Oxy-acids,  aromatic,  260 

in  the  urine,  260 

isolation  of,  201 

tests  for,  261 

<  >xy-amygdalic  acid  in  the  urine,  260 
/3-oxybutyric  acid,  94,  265 

relation  to  acetone,  265 
to  a-crotonic  acid,  265 
to  diacetic  acid,  265 
tests  for,  265 
Oxydases,  106 

in  the  liver,  402 

in  the  saliva,  1 12 

Oxyhsemocyanin,  329,  338 

(  (xvluemoglobin,  331 

estimation  of,  334 

isolation  of,  33.2 

properties  of,  333 

Oxy-nydroparacumaric  acid,  95 

<  txyneurin,  66 

<  >xypiperidin,  73 

<  >xyprotonic  acid,  38 

( >xyproto-8iil phonic  acid,  38 

PALMITIC  acid,  64,  93 
I'almitin.  6  I 
Pancreas,  405 

Kubnc'sdiv,  138 
Pancreatic  juice,  133 

amount  of,  135 

chemical  composition  of,  135 

ferments  of,  136 

general  properties  of,  1 34 

significance  of,  L33 

specific  gravity  of,  134 

zymogens  of,  136 
Parabanic  acid,  80,  82 
Paracasein,  1 12 
Paracholesterin,  67 
Paracresol,  89,  9C 


450 


INDEX. 


Paracresol  in  the  urine,  248 

Paraglobnlin,  314 

Paralactic  acid  in  the  urine,  269 

isolation  of,  269 
Paralbumin,  45 
Paranucleus,  43,  74,  77 
Para-oxy-benzoic  acid,  96 
Para-phenyl-acetic  acid,  95,  201 

in  the  urine,  260 

isolation  of,  207 
Para-oxy-phenyl-glycolic  acid,  95 

in  the  urine,  260 
Para-oxy-phenyl-lactic  acid,  95 

in  the  urine,  260 
Para-oxy-phenyl-propionic  acid,  95 

in  the  urine,  260 
Paraxanthin,  78,  240 
Paton's  globulin,  285,  289 
Penta-methylene-diamin,  74 
Pentoses,  54,  62 

in  the  urine,  283 

tests' for,  284 
Pepsin,  124,  126 

estimation  of,  129 
isolation  of,  127 
properties  of,  126 
Pepsinogen,  124 

estimation  of,  130 
test  for,  129 
Peptic  digestion,  products  of,  183 
Peptone-plasma,  313 
Peptones,  50,  169 
Pericardial  fluid,  analysis  of,  343 
Peritoneal  effusions,  analysis  of,  344 
Peroxy-jarotonic  acid.  38 
Pettenkofer' s  test,  147 
Phenaceturic  acid,  97,  259 

isolation  of,  259 

origin  of,  259 

properties  of,  259 
Phenol,  89,  96 

estimation  of,  in  the  urine,  249 
in  the  intestinal  contents,  200 
in  the  urine,  248 
tests  for,  201 
Phenyl-acetic  acid,  97 

in  the  intestinal  contents,  201 

isolation  of.  207 
Phenylhydrazin-test  for  sugar,  279 
Phenyl-propionic  acid,  97 

in  the  intestinal  contents,  201 

isolation  of,  207 
Phlebin,  327 
Phloretin,  25 
Phloridzin,  25 
Phosphates,  estimation  of.  in  the  urine, 

220 
Phospho-gluco-proteids,  44,  46 
Phosphor-carnic  acid,  351 
Photo-methsmoglobin,  336 
Phyllocyanic  acid,  22 
Phylloporphyrin,  22,  338 
Phylloxanth'in,  22 


Phymatorhusin,  297,  395 

Phytosterin,  67 

Pigments  of  the  blood,  328 

of  the  muscle-tissue,  353 

of  the  skin,  395 

of  the  urine,  291 

respiratory,  329 
Pine-apple  test  for  butyric  acid,  123 
Piperazin,  420 
Piria's  test  for  tyrosiri,  189 
Placenta,  431 
Plaques,  324 
Plasma,  albumose-,  313 

blood-,  312 

histon-,  326 

muscle-,  347 

oxalate-,  313 

peptone-,  313 

salt-,  313 
Plasminic  acid,  76,  77 
Plastin,  304 
Platner's  bile,  147 
Pleural  effusions,  analyses  of,  344 
Plosz'  urorubrin,  251 
Polysaccharides,  59 
Prseglobulin,  305 
Pro-enzymes,  101 
Propionic  acid,  93 
Protagon,  372 

isolation  of,  373 

properties  of,  372 
Protamins,  37,  69,  74 

in  the  spermatozoa,  421 
Proteids,  42 

digestion  of,  178 
Proteinochromes,  193 
Proteinochromogen,  193 
Proteins,  30 
Proteoses,  50 
Prothrombin,  319 
Proto-albumose,  50,  169 
Protons,  71 
Protophyllin,  25 
Pseudomucin,  45 
Pseudonucleins,  43,  74 
Ptomains,  91,  100 

acyclic,  91 

cyclic,  91 

in  the  intestinal  contents,  201 

in  the  urine,  299 
Ptyaliu,  110 

action  of,  110 

chemical  nature  of,  110 

isolation  of  111 
Ptyalinogen,  110 
Purin,  78 

bases,  43,  75,  77 
Pus,  analysis  of,  345 
Putrefaction,    analysis    of    products    of, 

206 
Putrescin,  73 

in  the  urine,  299 
Pyogenin,  372 


INDEX. 


451 


Pyosin,  372 
Pyrocatechin,  96 

in  the  urine,  '24s 
Pyrocatecnin-reaction  of  adrenal  glands, 

"435 
Pyrocatechuic  acid,  262 
Pyrocholesteric  acid,  152 

1  )  KXXIX  (see  also  Ohymosin),  130,  139 
xi     Reproductive  glands,  419 
Resorption  of  albumins,  172 

of  carbohydrates,  167 

of  fats,  181 
Reticulin,  383 
Retina,  379 

analysis  of,  380 

chromophanes  in,  381 

rhodopsin  in,  380 
Rhamnose,  284 
Rhodophane,  381 
Rhodopsin,  380 
Rosenbach's  reaction,  252 

SA.CCHARINIC  acid,  55,  91,  256 
Baccharo-lactonic  acid,  55 
Saccharose,  58 
Salamandrin,  398 
Salicin,  25 
Saligenin,  25 
Saliva,  108 

amount  of,  108 

analysis  of,  109,  110 

chemical  composition  of,  109 

chorda-,  109 

digestive  importance  of,  112 

extractives  in,  114 

ferments  in,  1 12 
s  in,  114 

general  characteristics  of,  108 

mineral  constituents  of,  114 

nitrite-  in,   1  ]:; 

paralytic,  109 
I  iuii  of,  108 

sulphocyanides  in,  1 13 

sympathetic,  LOfl 
Salivary  glands,  L08,  405 
Salkow-ki -  method  of  estimating  oxalic 
acid,  2  13 

holesterin,  163 
Salkowski-Volhard  method  of  estimating 
chlorides,  219 

Salinin,  69 

Salt-plasma,  813 

Saponification,  65 

s  1 1  •  i  n    iee  a  I-.,  Hypoxanthin 

le-i  for,  865 
Sarcylic  acid,  75 

nk'i  indirubin,  251 
Scherer't  red  pigment  of  tic  urine,  251 
■  tor  inosit,  860 
for  leucin,  188 
tor  tjrrosin,  I  39 
Schifl  i  uric  acid,  287 


Schmiedeberg's  histenzyme,  258 
Schiitzenberger's  hypothesis,  39 

Sclerotic,  377 
Scombrin,  69 
Scombron,  71 
Scyllite,  360 
Sebum,  397 
Semen,  419 

general  properties  of,  419 
Sericin,  47 

Serolin  in  the  feces,  208 
Serum  of  the  blood,  317 

composition  of,  317 
Serum-albumin,  41 

forms  of,  316 

in  the  urine,  288 

isolation  of,  316 

of  the  blood,  316 

test  for,  288 
Serum-casein,  314 
Serum-globulin,  41 

in  the  urine,  288 

isolation  of,  314 

properties  of,  315 

test  for,  288 
Siegfried's  carniferrin,  351 
Silicates  in  the  urine,  219 
Silurin,  70 
Skated,  90,  199 

isolation  of,  206 

tests  for,  200 
Skatol-carbonie  acid,  90 
isolation  of,  207 
test  for,  254 
Skatoxyl,  90 

sulphate  in  the  urine,  253 

tests  for,  254 
Skeletins,  47,  48,  389 
Skin,  394 

elei'din  granules  in,  394 

keratin  in,  394 
Smegma,  398 
Smith's  test,  159 
Soaps,  Ii5 
Solanidin,  26 
Solanin,  26 
Sorbite,  5  1 

Spectroscopic  lest  for  bilirubin,  159 
Spermanucleinic  acid,  75 
Spermatic  fluid,  420 
Spermatin,  120 
Spermatozoa,  421 

analysis  of,    121 

chemical  composition  of,  421 

protamine  in,  421 
Spermin,  420 

phosphate  of,  420 
Spirographin,  15 

Spleen.    IMC, 

arginin  in,  I  hi; 
Spongin,  17,  Is 
Spongioplasm,  308 
Starch* 


452 


INDEX. 


Steapsin,  138 

Stearic  acid,  64,  93 

Stearin,  64 

Stercobilin  in  the  feces,  207 

relation  to  urobilin,  292 
Stercorin  in  the  feces,  208 
Stoke' s  reagent,  330 
Stools  (see  also  Feces),  204 

acholic,  204 
Sturin,  69 
Succinic  acid,  95 
Sugar  in  lymph,  342 
Sulphates,  estimation  of,  in  the  urine,  220 
Sulphocyanides  in  the  gastric  juice,  132 

in  the  saliva,  113 

in  the  urine,  271 

test  for,  113 
Sulphur,  acid,  of  the  urine,  218 

neutral,  of  the  urine,  218 
Sulphur-test,  35 
Supporting  tissues,  382 
Suprarenin,  v.  Fiirth's,  436 
Sweat,  395 

analyses  of,  397 

chemical  composition  of,  396 

gases  of,  397 

general  characteristics  of,  396 

significance  of,  396 
Synaptose,  105 
Synovial  fluid,  345 
Synovin,  345 
Syntonin,  49,  169 

TAKTKONIC  acid,  83 
Tauriu,  88,  153 

in  muscle-tissue,  363 
isolation  of,  154 
Taurocarbaminic  acid,  88 
in  the  urine,  271 
Taurocholic  acid,  88,  149 
Teeth,  388 
Testicles,  419 

Tetra-hydro-dioxypyridin,  73 
Tetra-methylene-diamin,  73 
Theobromin,  78,  240 
Theophyllin,  78 
Thiosulphates  in  the  urine,  271 
Thrombin,  319 
Thrombosin,  320,  325 

-calcium,  320 
Thymin,  76 

Thyminic  acid,  76,  77,  323 
Thymo-nucleinic  acid,  75,  323 
Thymus  gland,  405 
Thyreoglobulin,  433 
Thvreo-nucleo-albumin,  434 
Thyroid  gland,  432 
Thyroiodine,  432 
Tissue-fibrinogen,  305 
Tollens'  orcin  test,  284- 

phloroglucin  test,  284 
Topfer's  method   of    estimating    hydro- 
chloric acid,  1 20 


Topfer's  test  for  hydrochloric  acid,  1 1 9, 1 20 
Toxalbumins,  100 
Toxins,  100 
Triglycerides,  64 
Trioxy-phenyl-propionic     acid    in    the 

urine,  260 
Trypsin,  136 

action  of,  137 

isolation  of,  137 

test  for,  137 
Trypsinogen,  136 
Tryptic  digestion,  174 

products  of,  185 
Tryptophan,  193 

tests  for,  193 
Tunicin,  389 
Tyrosin,  37,  86,  188 

in  the  urine,  270 

isolation  of,  189,  271 

tests  for,  189 
Tyrosin-hydantoinic  acid,  88 

UFFELMANN'S  test  for!acticacid,122 
Uramic  acids,  88 
Uramido- benzoic  acid,  88 
Urea,  estimation  of,  230 
formation  of,  86,  229 
in  the  urine,  222 
isolation  of,  from  the  urine,  230 
origin  of,  222 
properties  of,  227 
Urea-nitrate,  227 
Urea-oxalate,  228 
Ureids,  81 
Uric  acid,  80,  81 

estimation  of,  238 
in  the  urine,  233 
isolation  of,  237 
origin  of,  233 
properties  of,  236 
tests  for,  237 
Urine,  209 

acetone  in,  267 

acidity  of,  215 

albumins  in,  284 

allantoin  in,  243 

ammoniacal  decomposition  of,  210 

amount  of,  211 

aromatic  constituents  of,  247 

oxy-acids  of,  260 
carbohydrates  of,  275 
chemical  composition  of,  216 
chlorides  of,  216 
cholesterin  in,  298 
color  of,  210 

compound  glycocolls  in,  256 
conjugate  glucuronates  in,  255 

sulphates  in,  248 
cy stein  in,  272 
cystin  in,  273 
dextrin  in,  283 
diacetic  acid  in,  266 
fats  in,  298 


IXDEX. 


453 


Urine,  fatty  acids  in,  264 
ferments  in,  298 

gases  in.  299 

general  characteristics  of,  209 

glucose  in.  27»'> 

hippuric  acid  in.  257 

homogentisinic  acid  in.  261 

indican  in,  230 

inorganic  constituents  of,  216 

inosit  in.  262 

kreatinin  in,  244 

kynurenie  acid  in,  263 

lactose  in,  282 

lsevulose  in,  2*3 

lecithin  in,  298 

leucin  in,  270 

lithuric  acid  in,  263 

maltose  in,  283 

neutral  sulphur  in,  271 

nitrogenous  constituents  of,  222 

odor  of,  211 

organic  constituents  of,  221 

ornithuric  acid  in,  260 

oxalic  acid  in,  241 

oxaluric  acid  in,  241 
/i-oxybutyrie  acid  in,  265 
parallactic  acid  in,  269 

pentoses  in,  283 

phenaceturic  acid  in,  259 

phenols  in,  248 

phosphates  in,  217 

pigments  in,  291 

ptomains  in,  299 

reaction  of,  212 

skatol-carbonic  acid  in,  254 

skatoxyl  sulphate  in,  253 

specific  gravity  of,  212 

sulphates  in,  217 

tyrosin  in,  270 

urea  in,  222 

uric  acid  in,  233 

urocaninic  acid  in,  263 

uroltucinic  acid  in,  262 

volatile  fatly  acid-  in,  264 

xantbin-bases  in,  240 
I  Frobilin,  isolation  of,  293 

Jaffe's.  291,  293 

MacMuni, 
for,  293 
Urocaninic  acid.  263 
I  Irochloralic  acid,  2">"> 
I  frochrotne,  293 

isolation  of.  ■_! '. •  - 

I  fro<  rythrin,  2'.<1 
isolation  ol 

1'rofii-'  <,|i;iiiiatii 
.in  in,  261 

l.anialiii,   'J".  1 

for,  262 
rjroleucinic  acid  in  the  urine,  262 

I    ,  :..,•'.,  I 

L'roi  26 1 


Urorubin,  251 
Urorubrohsematin,  296 
Uroxanthinic  acid,  262 
Urrhodin,  251 
Urrhodinic  acid,  262 
Uterine  milk,  419 

VALERIANIC  acid,  93 
Vegetable  albumins.  41 

amyloid,  61 

globulins,  41 

gums,  60 
ViteUins,  42 
Vitellolutein,  429 
Vitellorubin,  429 
Y helioses,  50 
Vitreous  body,  379 

analysis  of,  379 

WALLEATH,  163 
Wang's     method     of     estimating 
indican,  253 
Weidel's  test  for  xanthin,  365 
Welcker's  method  of  estimating  the  total 

amount  of  blood,  309 
Wharton's  jelly,  382 
Whey,  acid,  416 

sweet,  408 
v.  Wittich's  method  of  isolating  pepsin, 
128  . 

XANTHIN,  78,  240 
in  muscle-tissue,  363 

isolation  of,  362 

properties  of,  364 

tests  for,  365 
Xantbin-bases,  43,  75,  77 

estimation  of,  241 

in  muscle-tissue,  363 

isolation  of,  363 

of  the  urine,  240 

origin  of,  241 
Xanthinic  leucomains,  79 
Xanthokreatinin,  84,  363 
Xanthophane,  :>sl 
Xanthoproteic  reaction,  34 

Xantliopsin,  3SO 

Xanthylic  acid,  75 
Xylose,  62,  284 

YEAST-NUCLEINIC  acid,  75 
folk  of  birds'  eggs,  426 
album inB  of,  127 
analysis  of,  127 
fats  of,  129 
bsematogen  in,  428 
lipochromes,  of,  429 
ovovitellin  in,  427 
white,  426 

Yolk    platelet-,    80,  42 


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large  and  handsome  8vo.  volume  of  1161  pages,  with  656  engravings.  Cloth,  $6 ;  leather,  $7. 

A  SYSTEM  OF  PRACTICAL  MEDICINE  BY  AMERICAN  AUTHORS.  Edited 
by  William  Pepper,  M.D.,  LL.D.  In  five  large  octavo  volumes,  containing  5573  pages 
and  198  illustrations.  Price  per  volume,  cloth,  $5 ;  leather,  $6 ;  half  Russia,  $7.  Sold 
by  subscription  only.     Prospectus  free  on  application  to  the  Publishers. 

A  PRACTICE  OF  OBSTETRICS  BY  AMERICAN  AUTHORS.  See  Jewett, 
page  9. 

ATTFIELD  (JOHN).  CHEMISTRY;  GENERAL,  MEDICAL  AND  PHAR- 
MACEUTICAL. New  (16th)  edition,  specially  revised  by  the  Author  for  America. 
In  one  handsome   12mo.  volume  of  784  pages,  with  88  illustrations.     Cloth,  $2.50,  net. 


Philadelphia,  706,  708  and  710  Sansom  St. — New  York,  111  Fifth  Avenue. 


LEA    BROTHERS    &     CO.'S    PUBLICATIONS. 


BACON  (GORHAM).  ON  THE  EAR.  New  (2d)  Edition.  One  12mo.  volume,  422 
pages,  with   1 14  engravings  and  3  colored  plates.     Cloth,  $2. 25,  net.     Just  ready. 

BALLENGER  (W.  L.)  AND  WIPPERN  (A.  G.).  A  POCKET  TEXT-BOOK 
OF  DISEASES  OF  THE  EYE,  EAR,  NOSE  AND  THROAT.  12mo.,  525 
pages,  with  148  illustrations,  and  6  colored  plates.  Cloth,  $2,  net.  Flexible  red  leather, 
|2.50,  n(t.     Just  ready. 

BARNES  (ROBERT  AND  FANCOURT).  A  SYSTEM  OF  OBSTETRIC  MED- 
ICINE  AND  SURGERY,  THEORETICAL  AND  CLINICAL.  The  Section  on 
Embryology  by  Prof.  Milnes  Marshaxl.  In  one  large  octavo  volume  of  872  pages, 
with  231  illustrations.     Cloth,  $5  ;  leather,  $6. 

B ARTHOLOW  ( ROBERT  S ) .  CHOLERA  ;  ITS  CA  USA  TION,  PRE  VENTION 
AND  TREATMENT.  In  one  12mo.  volume  of  127  pages,  with  9  illustrations 
Cloth,  $1.25. 

BILLINGS  (JOHN  S.).  THE  NATIONAL  MEDICAL  DICTIONARY.  Includ- 
ing in  one  alphabet  English,  French,  German,  Italian  and  Latin  Technical  Terms  used  in 
Medicine  and  the  Collateral  Sciences.  In  two  very  handsome  imperial  octavo  volumes, 
containing  1574  pages  and  two  colored  plates.  Per  volume,  cloth,  $6  ;  leather,  $7  ;  half 
Morocco,  $8.50.     For  sale  by  subscription  only.     Specimen  pages  on  application. 

BLACK  (D.  CAMPBELL).  THE  URINE  IN  HEALTH  AND  DISEASE, 
AND  URINARY  ANALYSIS,  PHYSIOLOGICALLY  AND  PATHOLOGI- 
CALLY CONSIDERED.  In  one  12mo.  volume  of  256  pages,  with  73  engravings. 
Cloth,  $2.75. 

BLOXAM  (C.  L.).  CHEMISTRY,  INORGANIC  AND  ORGANIC.  With 
Experiments.  New  American  from  the  fifth  London  edition.  In  one  handsome  octavo 
volume  of  727  pages,  with  292  illustrations.     Cloth,  $2 ;  leather,  $3. 

BROCKWAY  (FRED.  J.).  A  POCKET  TEXT-BOOK  OF  ANATOMY.  12mo. 
of  about  400  pages,  richly  illustrated.     Shortly. 

BRUCE  (J.  MITCHELL).  MATERIA  MEDIC  A  AND  THERAPEUTICS. 
Js'ew  (6th)  edtion.  In  one  12mo.  volume  of  600  pages.  Just  ready.  Cloth,  $1.50,  net. 
See  Students'  Series  of  Manuals,  page  14. 

PRINCIPLES  OF  TREATMENT.     In  one  octavo  volume  of  625  pages.     Just 


Ready.     Cloth,  $3.75,  net. 

BRYANT  < THOMAS).  THE  PRACTICE  OF  SURGERY.  Fourth  American 
from  the  fourth  English  edition.  In  one  imperial  octavo  volume  of  1040  pages,  with  727 
illustrations.     Cloth,  $6.50;  leather,  $7.50. 

BURCHARD  (HENRY  H.).  DENTAL  PATHOLOGY  AND  THERAPEUTICS, 
INCLUDING  PHARMACOLOGY.  Handsome  octavo,  575  pages,  with  400  illus- 
trations.    Cloth,  $5  ;  leather,  $6,  net. 

BURNETT    (CHARLES  H.).     THE  EAR:   ITS  ANATOMY,  PHYSIOLOGY 

AND  DISEASES.     A  Practical  Treatise  for  the  Use  of  Students  and  Practitioners. 
Second  edition.     8vo.,  580  pages,  with  107  illustrations.     Cloth,  $4 ;  leather,  $5. 

CARTER  (R.  BRUDENELL)  AND  FROST  (W.  ADAMS).  OPHTHALMIC 
SI' lie,  FRY.  In  one  pocket-size  12mo.  volume  of  559  pages,  with  91  engravings  and 
one  plate,     ('loth,  $2.25.     See  Series  of  Clinical  Manuals,  page  13. 

CASPARI  I  CHARLES,  JR.).  A  TREAT ISE  ON  PHARMACY.  For  Students 
and  Pharmacists.  In  one  handsome  octavo  volume  of  680  pages,  with  288  illustrations. 
Cloth,  $4.60. 

CHAPMAN  i  HENRY  C).  A  TREATISE  ON  HUMAN  PHYSIO  LOGY.  New 
(2d)  edition.  In  one  octavo  volume  of  '.'21  pages,  with  595  illustrations.  Just  ready. 
Cloth,  $4.26;   leather,  f5.26,  net. 

Philadelphia,  706,  708  and  710  Sansom  St.— New  York,  111  Fifth  Avenue. 


4  LEA    BROTHERS    &     CO.'S    PUBLICATIONS. 

CHARLES  (T.  CRANSTOUN).  THE  ELEMENTS  OF  PHYSIOLOGICAL 
AND  PATHOLOGICAL  CHEMISTRY.  In  one  handsome  octavo  volume  of  451 
pages,  with  38  engravings  and  1  colored  plate.     Cloth,  $3.50. 

CHEYNE  (W.  WATSON).  THE  TREATMENT  OF  WOUNDS,  ULCERS 
AND  ABSCESSES.    In  one  12mo.  volume  of  207  pages.     Cloth,  $1.25. 

CHEYNE  (W.  WATSON)  AND  BURGHARD  (F.  P.).  SURGICAL  TREAT- 
MENT. In  seven  octavo  volumes,  illustrated.  Volume  I,  just  ready.  299  pages  and 
66  engravings.  Cloth,  $3.00,  net.  Volume  II,  just  ready.  382  pages,  141  engravings. 
Cloth,  $4.00,  net.  Volume  III.  Just  Ready.  300  pages,  100  engravings.  Cloth,  $3.50,  net. 
Vol.  IV.     In  Press. 

CLARKE  (W.  B.)  AND  LOCKWOOD  (C.  B.).  THE  DISSECTOR'S  MANUAL. 
In  one  12mo.  volume  of  396  pages,  with  49  engravings.  Cloth,  $1.50.  See  Students'  Series 
of  Manuals,  page  14. 

CLELAND  (JOHN).  A  DIRECTORY  FOR  THE  DISSECTION  OF  THE 
HUMAN  BODY.     In  one  12mo.  volume  of  178  pages.     Cloth,  $1.25. 

CLINICAL  MANUALS.     See  Series  of  Clinical  Manuals,  page  13. 

CLOUSTON  (THOMAS  S.).  CLINICAL  LECTURES  ON  MENTAL  DIS- 
EASES. New  (5th)  edition.  Crown  8vo.,  of  736  pages  with  19  colored  plates.  Cloth, 
$4.25,  net. 

S^°  Folsom's  Abstract  of  Laivs  of  U.  S.  on  Custody  of  Insane,  octavo,  $1.50,  is  sold  in 
conjunction  with  Clouston  on  Mental  Diseases  for  $5. 00,  net,  for  the  two  works. 

CLOWES    (FRANK).  AN  ELEMENTARY    TREATISE    ON  PRACTICAL 

CHEMISTRY  AND  QUALITATIVE  INORGANIC  ANALYSIS.    From  the 

fourth  English  edition.  In  one  handsome  12mo.  volume  of  387  pages,  with  55  engrav- 
ings.    Cloth,  $2.50. 

COAKLEY  (CORNELIUS  G.).  THE  DIAGNOSIS  AND  TREATMEN1  OF 
DISEASES  OF  THE  NOSE,  THROAT,  NASO-PHARYNX  AND  TRACHEA. 
In  one  12mo.  volume  of  526  pages,  with  92  engravings,  and  2  colored  plates. 

COATES  (W.  E.,  Jr.).  A  POCKET  TEXT-BOOK  OF  BACTERIOLOGY 
AND  HYGIENE.     12mo.,  of  about  350  pages  with  many  illustrations.     Shortly. 

COATS  (JOSEPH).  A  TREATISE  ON  PATHOLOGY.  In  one  volume  of  829 
pages,  with  339  engravings.     Cloth,  $5.50 ;  leather,  $6.50. 

COLEMAN  (ALFRED).  A  MANUAL  OF  DENTAL  SURGERY  AND  PATH- 
OLOGY. With  Notes  and  Additions  to  adapt  it  to  American  Practice.  By  Thos.  C. 
Stellwagen,  M.A.,  M.D.,  D.D.S.  In  one  handsome  octavo  volume  of  412  pages,  with 
331  engravings.     Cloth,  $3.25. 

COLLINS  (C.  P.).  A  POCKET  TEXT-BOOK  OF  MEDICAL  DIAGNOSIS. 
12mo.  of  about  350  pages.     Shortly. 

COLLINS  (H.  D.)  AND  ROCKWELL  (W.  H.,  JR.).  A  POCKET  TEXT- 
BOOK OF  PHYSIOLOGY.  12mo.,  of  316  pages,  with  153  illustrations.  Just 
Ready.     Cloth,  $1.50,  net;  flexible  red  leather,  $2.00,  net. 

CONDIE  (D.  FRANCIS).  A  PRACTICAL  TREATISE  ON  THE  DISEASES 
OF  CHILDREN.     Sixth  edition.     8vo.  719  pages.     Cloth,  $5.25 ;  leather,  $6.25. 

CORNIL  (V.).  SYPHILIS:  ITS  MORBID  ANATOMY,  DIAGNOSIS  AND 
TREATMENT.  Translated,  with  Notes  and  Additions,  by  J.  Henry  C.  Simes,  M.D., 
and  J.  William  White,  M.D.  In  one  8vo.  volume  of  461  pages,  with  84  illustrations. 
Cloth,  $3.75. 


Philadelphia,  706,  708  and  710  Sansom  St.— New  York,  111  Fifth  Avenue. 


LEA    BROTHERS    &     CO.'S    PUBLICATIONS. 


CROCKETT  (M.  A.).  A  POCKET  TEXT-BOOK  OF  DISEASES  OF 
WOMEN.  12mo.  of  368  pages,  with  107  illustrations.  Just  Beady.  Cloth,  $1.50,  net. 
Flexible  Red  Leather,  §2.00,  net. 

CROOK  (JAMES  K.).     MINERAL  WATEBS  OF  UNITED  STATES.     Octavo 
574  pages.     Just  ready.     Cloth,  $3.50,  net. 

CULBRETH  (DAVID  M.  R.).    MATEBIA  MEDICA  AND  PHABMACOLOQY. 

New  ( 2d)  edition.     In  one  handsome  octavo  volume  of  881  pages,  with  464  engravings. 
Cloth,  $4.50,  net.     Just  Ready. 

CUSHNY  (ARTHUR  R.)  A  TEXT-BOOK  OF  PHABMACOLOQY  AND 
THEBAPEUTICS.     Octavo  of  728  pages,  with  47  illustrations.     Cloth,  $3.75,  net. 

DALTON  (JOHN  C).  A  TBEATISE  ON  HUMAN  PHYSIOLOGY.  Seventh 
edition, thoroughly  revised.  Octavo  of  722  pages, with  252  engravings.  Cloth,  $5;  leather,$6. 

DOCTBINES  OF  THE  CIBCULATION  OF  THE  BLOOD.    In  one  hand- 


some 12mo.  volume  of  293  pages.     Cloth,  $2. 

DAVENPORT  (F.  H.).  DISEASES  OF  WOMEN.  A  Manual  of  Gynecology. 
For  the  use  of  Students  and  General  Practitioners.  New  (3d)  edition.  In  one  hand- 
some 12mo.  volume,  387  pages  and  150  engravings.     Cloth,  $1.75,  net. 

DAVIS  (F.H.).    LECTUBES  ON  CLINICAL  MEDICINE.    Second  edition.    In 

one  12mo.  volume  of  287  pages.     Cloth,  $1.75. 

DAVIS  (EDWARD  P.).  A  TBEATISE  ON  OBSTETBICS.  For  Students  and 
Practitioners.  In  one  very  handsome  octavo  volume  of  546  pages,  with  217  engravings, 
and  30  full-page  plates  in  colors  and  monochrome.     Cloth,  $5 ;  leather,  $6. 

DE  LA  BECHE'S  GEOLOGICAL  OBSEBVEB.  In  one  large  octavo  volume  of  700 
pages,  with  300  engravings.     Cloth,  $4. 

DENNIS  (FREDERIC  S.)  AND  BILLINGS  (JOHN  S.).  A  SYSTEM  OF 
SUBGEBY.  In  Contributions  by  American  Authors.  In  four  very  handsome  octavo 
volumes,  containing  3652  pages,  with  1585  engravings,  and  45  full-page  plates  in  colors 
and  monochrome.  Per  volume,  cloth,  $6 ;  leather,  $7 ;  half  Morocco,  gilt  back  and 
top,  $8.50.     For  sale  by  subscription  only.     Full  prospectus  free. 

DERCUM  (FRANCIS  X.),  Editor.  A  TEXT-BOOK  ON  NEBVOUS  DIS- 
EASES. By  American  Authors.  In  one  handsome  octavo  volume  of  1054  pages,  with 
341  engravings  and  7  colored  plates.     Cloth,  $6 ;  leather,  $7,  net. 

DE  SCHWEINITZ  (GEORGE  E.).  THE  TOXIC  AMBLYOPIAS;  THEIB 
CLASSIFICATION,  HIS  TOBY,  SYMPTOMS,  PATHOLOGY  AND  TBEAT- 
MENT.  Very  handsome  octavo,  240  pages,  46  engravings,  and  9  full-page  plates  in 
colors.     Limited  edition,  de  luxe  binding,  $4,  net. 

DRAPER  ( JOHN  C . ) .  MEDICA L  PHYSICS.  A  Text-book  for  Students  and  Prac- 
titioners oi  Medicine.     Octavo  of  734  pages,  with  376  engravings.     Cloth,  $4. 

DRUITT  (ROBERT).  THE  PBINCIPLES  AND  PBACTICE  OF  MODEBN 
8URGER  )'.  A  new  American,  from  the  twelfth  London  edition,  edited  by  Stanley 
Boyd,  F.R.(  .S.     Large  octavo,  965  pages,  with  373  engravings.     Cloth,  $4;  leather,  $5. 

DUANE  (ALEXANDER).  A  DICTIONARY  OF  MEDICINE  AND  THE 
ALLIED  SCIENCES.  Comprising  the  Pronunciation,  Derivation  and  Full  Explan- 
ation of  Medical,  Dental,  Pharmaceutical  and  Veterinary  Terms.  Together  with  much 
Collateral  Descriptive  Matter,  Numerous  Tables,  etc.  New  (3d)  edition.  Square  octavo 
volume  of  852  pages  with  8  colored  plates.  Just  Ready.  Cloth,  $3.00,  net;  limp 
leather,  $4.00,  net. 

DUDLEY  'E.  C.).  A  TREATISE  ON  THE  PBINCIPLES  AND  PBACTICE 
OF  GYNECOLOGY.  For  Students  and  Practitioners.  New  (2d)  edition.  In  one 
very  handsome  octavo  volume  of  717  pages,  with  453  engravings,  of  which  47  are 
colored,  and  -  full  page  plate-,  in  colors  and  monochrome.  Just  Ready.  Cloth, $5.00,  net; 
leather,  $6.00,  net;  liall  morocco,  $6.50,  net. 

DUNCAN  <J.  MATTHEWS).  CLINICAL  LECTUBES  ON  THE  DISEASES 
OF  WOMEN.  Delivered  in  St.  Bartholomew's  Hospital.  In  one  octavo  volume  of 
176  pages.    Cloth,  $1.60. 

Philadelphia,  706,  708  and  710  Sansom  St.— New  York,  111  Fifth  Avenue. 


LEA    BROTHERS    &     C  0 .'  S    PUBLICATIONS. 


DUNGLISON  (EOBLEY).  A  DICTIONARY  OF  MEDICAL  SCIENCE.  Con- 
taining a  full  Explanation  of  the  Various  Subjects  and  Terms  of  Anatomy,  Physiology, 
Medical  Chemistry,  Pharmacy,  Pharmacology,  Therapeutics,  Medicine,  Hygiene,  Dietetics. 
Pathology,  Surgery,  Ophthalmology,  Otology,  Laryngology,  Dermatology,  Gynecology, 
Obstetrics,  Pediatrics,  Medical  Jurisprudence,  Dentistry,  Veterinary  Science,  etc.,  etc. 
By  Robley  Dunqlison,  M.D.,  LL.D.,  late  Professor  of  Institutes  of  Medicine  in  the 
Jefferson  Medical  College  of  Philadelphia.  Edited  by  Bichard  J.  Dtjnglison,  A.M., 
M.D.  Twenty-second  edition,  thoroughly  revised  and  greatly  enlarged  and  improved, 
with  the  Pronunciation,  Accentuation  and  Derivation  of  the  Terms.  With  Appendix. 
Imperial  octavo  of  1350  pages,  with  thumb  letter  index.  Just  ready.  Cloth,  $7.00,  net  - 
leather,  $8.00,  net.     This  edition  contains  portrait  of  Dr.  Robley  Dunglison. 

DUNHAM  (EDWARD  K.).  MORBID  AND  NORMAL  HISTOLOGY.  Octavo, 
450  pages,  with  360  illustrations.     Cloth,  $3. 25,  net. 

NORMAL  HISTOLOGY.      New   (2d)   edition.     Octavo,  319  pages,  with  244 

illustrations.     Just  Ready.     Cloth,  $2.50,  net. 

EDES  (ROBERT  T.).  TEXT-ROOK  OF  THERAPEUTICS  AND  MATERIA 
MEDIC  A.     In  one  8vo.  volume  of  544  pages.     Cloth,  $3.50 ;  leather,  $4.50. 

EDIS  (ARTHUR  W.).  DISEASES  OF  WOMEN.  A  Manual  for  Studentsand 
Practitioners.  In  one  handsome  8vo.  volume  of  576  pages,  with  148  engravings. 
Cloth,  $3 ;  leather,  $4. 

EGBERT  (SENECA).  HYGIENE  AND  SANITATION.  In  one  12mo.  volume  of 
359  pages,  with  63  illustrations. 

ELLIS  (GEORGE  VINER).  DEMONSTRATIONS  IN  ANATOMY.  Being  a 
Guide  to  the  Knowledge  of  the  Human  Body  by  Dissection.  From  the  eighth  and  revised 
English  edition.     Octavo,  716  pages,  with  249  engravings.     Cloth,  $4.25 ;  leather,  $5.25. 

EMMET    (THOMAS   ADDIS).     THE   PRINCIPLES  AND   PRACTICE    OF 

G  YNJECOL  OGY.  For  the  use  of  Students  and  Practitioners.  Third  edition,  enlarged 
and  revised.     8vo.  of  880  pages,  with  150  original  engravings.     Cloth,  $5 ;  leather,  $6. 

ERICHSEN  (JOHN  E.).  THE  SCIENCE  AND  ART  OF  SURGERY.  A  new 
American  from  the  eighth  enlarged  and  revised  London  edition.  In  two  large  octavo 
volumes  containing  2316  pages,  with  984  engravings.     Cloth,  $9 ;  leather,  $11. 

ESSIG  (CHARLES  J.).  PROSTHETIC  DENTISTRY.  New  (2d)  Edition.  See 
American  Text-books  of  Dentistry,  page  2. 

EVANS  (DAVID  J.).  A  POCKET  TEXT-BOOK  OF  OBSTETRICS.  12mo. 
of  409  pages,  with  148  illustrations.  Cloth,  $1.75,  net;  limp  leather,  $2.25,  net.  Justready. 

EWING  (JAMES).  A  PRACTICAL  TREATISE  ON  THE  BLOOD.  Hand- 
some octavo  of  about  600  pages,  with  many  illustrations.     Preparing. 

FARQUHARSON  (ROBERT).  A  GUIDE  TO  THERAPEUTICS.  Fourth 
American  from  fourth  English  edition,  revised  by  Frank  Woodbury,  M.D.  In  one 
12mo.  volume  of  581  pages.     Cloth,  $2.50. 

FIELD  (GEORGE  P.).  A  MANUAL  OF  DISEASES  OF  THE  EAR.  Fourth 
edition.     Octavo,  391  pages,  with  73  engravings  and  21  colored  plates.     Cloth,  $3.75. 

FLINT  (AUSTIN).  A  TREATISE  ON  THE  PRINCIPLES  AND  PRACTICE 
OF  MEDICINE.  New  (7th)  edition,  thoroughly  revised  by  Frederick  P.  Henry, 
M.D.     In  one  large  8vo.  volume  of  1143  pages,  with  engravings.     Cloth,  $5 ;  leather,  $6. 

A  MANUAL  OF  AUSCULTATION  AND  PERCUSSION;  of  the  Physi- 
cal Diagnosis  of  Diseases  of  the  Lungs  and  Heart,  and  of  Thoracic  Aneurism.  Fifth 
edition,  revised  by  James  C.  Wilson,  M.D.  In  one  handsome  12mo.  volume  of  274 
pages,  with  12  engravings. 

A  PRACTICAL  TREATISE  ON  THE  DIAGNOSIS  AND  TREAT- 
MENT OF  DISEASES  OF  THE  HEART.  Second  edition,  enlarged.  In  one 
octavo  volume  of  550  pages.     Cloth,  $4. 

A   PRACTICAL   TREATISE  ON  THE  PHYSICAL  EXPLORATION 

OF  THE  CHEST,  AND  THE  DIAGNOSIS  OF  DISEASES  AFFECTING 
THE  RESPIRATORY  ORGANS.  Second  and  revised  edition.  In  one  octavo  vol- 
ume of  591  pages.     Cloth,  $4.50. 

Philadelphia,  706,  708  and  710  Sansom  St. — New  York,  111  Fifth  Avenue. 


LEA    BROTHERS    &     CO.'S    PUBLICATIONS. 


MEDICAL  ESSA  YS.     In  one  12mo.  volume  of  210  pages.     Cloth,  $1.38. 

ON  PHTHISIS:  ITS  MORBID  ANATOMY,  ETIOLOGY,  ETC.    A  Serie6 

of  Clinical  Lectures.     In  one  8vo.  volume  of  442  pages.     Cloth,  $3.50. 

FOLSOM  (C.  F.).  AN  ABSTRACT  OF  STATUTES  OF  U.  S.  ON  CUSTODY 
OF  THE  INSANE.  In  one  8vo.  volume  of  108  pages.  Cloth,  $1.50.  With  Clouston 
on  Mental  Diseases  (see  page  4),  at  $5.00,  net,  for  the  two  works. 

FORMULARY,  THE  NATIONAL.  See  Stille,  Maisch  &  Caspars  National  Dispensa- 
tory, page  14. 

FORMULARY,  POCKET.     See  page  1. 

FOSTER  (MICHAEL).  A  TEXT-BOOK  OF  PHYSIOLOGY.  New  (6th)  and 
revised  American  from  the  sixth  English  edition.  In  one  large  octavo  volume  of  923 
pages,  with  257  illustrations.     Cloth,  $4.50 ;  leather,  $5.50. 

FOTHERGILL    (J.    MILNER).     THE  PRACTITIONERS  HAND-BOOK   OF 
TREATMENT.     Third   edition.     In   one   handsome   octavo   volume   of    664   nases 
Cloth,  $3.75 ;  leather,  $4.75.  ' 

FOWNES  (GEORGE).  A  MANUAL  OF  ELEMENTARY  CHEMISTRY  (IN- 
ORGANIC AND  ORGANIC).  Twelfth  edition.  Embodying  Watts'  Physical  and 
Inorganic  Chemistry.  In  one  royal  12mo.  volume  of  1061  pages,  with  168  engravings,  and 
1  colored  plate.     Cloth,  $2.75 ;  leather,  $3.25.  ** 

FRANKLAND  (E.)  AND  JAPP  (F.  R.).  INORGANIC  CHEMISTRY.  In  one 
handsome  octavo  volume  of  677  pages,  with  51  engravings  and  2  plates.  Cloth  $3  75  • 
leather,  $4.75. 

FULLER   (EUGENE).    DISORDERS  OF  THE  SEXUAL  ORGANS  IN  THE 

MALE.     In  one  very  handsome  octavo  volume  of  238  pages,  with  25  engravings  and 
8  full-page  plates.     Cloth,  $2. 

FULLER  (HENRY) .  ON  DISEASES  OF  THE  L  UNGS  AND  AIR-PASS  A  GES. 
Their  Pathology,  Physical  Diagnosis,  Symptoms  and  Treatment.  From  second  English 
edition.     In  one  8vo.  volume  of  475  pages.     Cloth,  $3.50. 

GALLAUDET  (BERN  B.).  A  POCKET  TEXT-BOOK  ON  SURGERY. 
12mo.  of  about  400  pages,  with  many  illustrations.     Shortly. 

GANT  ( FREDERICK  JAMES ) .  THE  STUDENT'S  S UR GER  Y.  A  Multum  in 
Parvo.     In  one  square  octavo  volume  of  845  pages,  with  159  engravings.     Cloth,  $3.75. 

GERRISH  (FREDERIC  H.).  A  TEXT-BOOK  OF  ANATOMY.  By  American 
Authors.  Edited  by  Frederic  H.  Gerrish,  M.D.  In  one  imp.  octavo  volume  of  915 
pages,  with  950  illustrations  in  black  and  colors.  Cloth,  $6.50;  flexible  water-proof, 
$7  ;  sheep,  $7.50,  net. 

GIBBES    (HENEAGE).    PRACTICAL   PATHOLOGY  AND   MORBID   HIS- 

TOLOG  Y.   Octavo  of  314  pages,  with  60 illustrations,  mostly  photographic.   Cloth,  $2.75. 

GOULD  (A.  PEARCE).  SURGICAL  DIAGNOSIS.  In  one  12mo.  volume  of  589 
pages.     Cloth,  $2.     See  Students'  Series  of  Manuals,  page  14. 

GRAY  (HENRY).  ANATOMY,  DESCRIPTIVE  AND  SURGICAL.  New 
American  edition  thoroughly  revised.  In  one  imperial  octavo  volume  of  1239  pages, 
with  772  large  and  elaborate  engravings.  Price  with  illustrations  in  colors,  cloth,  $7 ; 
leather,  $8.     Price,  with  illustrations  in  black,  cloth,  $6 ;  leather,  $7. 

GREEN  T.  HENRY).  AN  INTRODUCTION  TO  PATHOLOGY  AND  MOR- 
BII)  ANATOMY.  New  (9th)  American  from  ninth  and  revised  English  edition. 
Oct.  666  page*,  with  339  engravings  and  4  colored  plates.     Cloth,  $3.25,  net.     Just  ready. 

GREENE  (WILLIAM  H.).    A  MANUAL  OF  MEDICAL  CHEMISTRY.    For 

the  Use  of  Students.     Based  upon  Bowman's  Medical  Chemistry.     In  one  12mo.  volume 
of  310  pages,  with  74  illustrations.     Cloth,  $1.75. 

GRINDON  (JOSEPH).     A    POCKET   TEXT-BOOK  OF  SKIN  DISEASES. 

J  I'm'),  of   350  pages,  with  many  illustrations.      Shortly. 

GROSS  (SAMUEL  D.).  A  PRACTICAL  TREATISE  ON  THE  DISEASES, 
INJURIES  AND  MALFORMATIONS  OF  THE  URINARY  BLADDER, 
HIE  I -HOST ATE  a  LAN  I)  AND  THE  URETHRA.  Third  edition,  revised  by 
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HABERSHON  (S.  0.).  ON  THE  DISEASES  OF  THE  ABDOMEN,  comprising 
those  of  the  Stomach,  (Esophagus,  Caecum,  Intestines  and  Peritoneum.  Second  Amer- 
ican from  the  third  English  edition.  In  one  octavo  volume  of  554  pages,  with  11  engrav- 
ings.    Cloth,  $3.50. 

HALL  (WINFIELD  S.).  TEXT-BOOK  OF  PHYSIOLOGY.  Octavo,  672  pages, 
with  343  engravings  and  6  colored  plates.     Cloth,  $4.00,  net;    leather,  $5.00,  net. 

HAMILTON  ( ALLAN  McLANE ) .  NEB  VO  US  DISEASES,  THEIB  DESCBIP- 
TION  AND  TBEA  TMENT.  Second  and  revised  edition.  In  one  octavo  volume  of 
598  pages,  with  72  engravings.     Cloth,  $4. 

HARDAWAY  (W.  A.).  MANUAL  OF  SKIN  DISEASES.  New  (2d)  edition. 
12mo.,  560  pages  with  40  illustrations  and  2  colored  plates.     Cloth,  $2.25,  net. 

HARE  (HOBART  AMORY).  A  TEXT-BOOK  OF  PB ACTIO AL  THEBA- 
PE  UTICS,  with  Special  Reference  to  the  Application  of  Remedial  Measures  to  Disease 
and  their  Employment  upon  a  Rational  Basis.  With  articles  on  various  subjects  by  well- 
known  specialists.  New  (8th)  and  revised  edition.  In  one  octavo  volume  of  796  pages, 
with  37  engravings  and  3  colored  plates.    Just  Beady.  Cloth,  $4. 00,  net ;  leather,  $5. 00,  net. 


PBACTICAL  DIAGNOSIS.  The  Use  of  Symptoms  in  the  Diagnosis  of  Dis- 
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with  205  engravings,  and  14  full-page  plates.     Cloth,  $5,  net. 

Editor.     A  SYSTEM  OF  PBACTICAL  THEBAPEUTICS.     By  American 


and  Foreign  Authors.  In  a  series  of  contributions  by  eminent  practitioners.  In  four 
large  octavo  volumes  comprising  4600  pages,  with  476  engravings.  Vol.  IV.,  now  ready. 
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ON  THE  MEDICAL  COMPLICATIONS  AND  SEQUELS  OF  TYPHOID 

FEVER.    Octavo,  276  pages,  21  engravings,  and2  full-page  plates.     Cloth,  $2.40,  net. 

HARTSHORNE  (HENRY).  ESSENTIALS  OF  THE  PBINCIPLES  AND 
PBACTICE  OF  MEDICINE.  Fifth  edition.  In  one  12mo.  volume,  669  pages, 
with  144  engravings.     Cloth,  $2.75 ;  half  bound,  $3. 

A   HANDBOOK  OF  ANATOMY  AND   PHYSIOLOGY.     In  one  12mo. 


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A  CONSPECTUS  OF  THE  MEDICAL  SCIENCES.     Comprising  Manuals 


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Obstetrics.  Second  edition.  In  one  royal  12mo.  volume  of  1028  pages,  with  477  illus- 
trations.    Cloth,  $4.25;  leather,  $5. 

HAYDEN  (JAMES  R.).  A  MANUAL  OF  VENEREAL  DISEASES.  New  (2d) 
edition.     In  one  12mo.  volume  of  304  pages,  with  54  engravings.     Cloth,  $1.50,  net. 

HAYEM   (GEORGES)  AND  HARE   (H.  A.).    PHYSICAL  AND  NATURAL 

THERAPEUTICS.  The  Remedial  Use  of  Heat,  Electricity,  Modifications  of  Atmos- 
pheric Pressure,  Climates  and  Mineral  Waters.  Edited  by  Prof.  H.  A.  Hare,  M.D. 
In  one  octavo  volume  of  414  pages,  with  113  engravings.     Cloth,  $3. 

HERMAN  (G.  ERNEST).  FIRST  LINES  IN  MIDWIFERY.  12mo.,  198  pages, 
with  80  engravings.     Cloth,  $1.25.     See  Students1  Series  of  Manuals,  page  14. 

HERMANN  (L.).  EXPERIMENTAL  PHARMACOLOGY.  A  Handbook  of  the 
Methods  for  Determining  the  Physiological  Actions  of  Drugs.  Translated  by  Robert 
Meape  Smith,  M.D.     In  one  12mo.  vol.  of  199  pages,  with  32  engravings.     Cloth,  $1.50. 

HERRICK  (JAMES  B.).  A  HANDBOOK  OF  DIAGNOSIS.  In  one  handsome 
12mo.   volume  of  429  pages,  with  80  engravings  and  2  colored  plates.     Cloth,  $2.50. 

HILL  (BERKELEY).  SYPHILIS  AND  LOCAL  CONTAGIOUS DISOBDEBS. 
In  one  8vo.  volume  of  479  pages.     Cloth,  $3.25. 

HILLIER  (THOMAS).  A  HANDBOOK  OF  SKIN  DISEASES.  Second  edition. 
In  one  royal  12mo.  volume  of  353  pages,  with  two  plates.     Cloth,  $2.25. 


Philadelphia,  706.  708  and  710  Sansom  St.— New  York,  111  Fifth  Avenue. 


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HIRST  (BARTON  C.)  AND  PIERSOL  (GEORGE  A.).  HUMAN  MONSTROS- 
ITIES. Magnificent  folio,  containing  220  pages  of  text  and  illustrated  with  123  engrav- 
ings and  39  large  photographic  plates  from  nature.  In  four  parts,  price  each,  $5.  Limited 
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HOBLYN  (RICHARD  D.).  A  DICTIONARY  OF  THE  TERMS  USED  IN 
MEDICINE  AND  THE  COLLATERAL  SCIENCES.  New  (13th)  edition.  In 
one  12mo.  volume  of  845  pages.     Just  Ready.     Cloth,  $3.00,  net. 

HODGE  ( HUGH  L. ) .  ON  DISEASES  PEC ULIAR  TO  WOMEN,  INCL  UDING 
DISPLACEMENTS  OF  THE  UTERUS.  Second  and  revised  edition.  In  one 
8vo.  volume  of  519  pages,  with  illustrations.     Cloth,  $4.50. 

HOFFMANN  (FREDERICK)  AND  POWER  (FREDERICK  B.).  A  MANUAL 
OF  CHEMICAL  ANALYSIS,  as  Applied  to  the  Examination  of  Medicinal  Chemicals 
and  their  Preparations.  Third  edition,  entirely  rewritten  and  much  enlarged.  In  one 
handsome  octavo  volume  of  621  pages,  with  179  engravings.     Cloth,  $4.25. 

HOLMES  (TIMOTHY).  A  TREATISE  ON  SURGERY.  Its  Principles  and 
Practice.  A  new  American  from  the  fifth  English  edition.  Edited  by  T.  Pickering 
Pick,  F.R.C.S.     Octavo,  1008  pages,  with  428  engravings.     Cloth,  $6;  leather,  $7. 

A  SYSTEM  OF  SURGERY.     With  notes  and  additions  by  various  American 


authors.  Edited  by  John  H.  Packard,  M.  D.  In  three  very  handsome  8vo.  volumes 
containing  3137  double-columned  pages,  with  979  engravings  and  13  lithographic  plates. 
Per  volume,  cloth,  $6  ;  leather,  $7  ;  half  Russia.  $7.50.     For  sale  by  subscription  only. 

HORNER  (WILLIAM  E.).  SPECIAL  ANATOMY  AND  HISTOLOGY.  Eighth 
edition,  revised  and  modified.  In  two  large  8vo.  volumes  of  1007  pages,  containing  320 
engravings.     Cloth,  $6. 

HUDSON  (A.).  LECTURES  ON  THE  STUDY  OF  FEVER.  In  one  octavo 
volume  of  308  pages.     Cloth,  $2.50. 

HYDE  f  JAMES  NEVINS).  A  PRACTICAL  TREATISE  ON  DISEASES  OF 
THE  SKIN.  New  (5th)  edition,  thoroughly  revised.  Octavo,  866  pages,  with  111 
engravings  and  24  full-page  plates,  8  of  which  are  colored.  Cloth,  $4.60,  net;  leather, 
$6.50,  net;  half  morocco,  $6.00,  net. 

JACKSON  <  GEORGE  THOMAS ) .  THE  READ  Y-REFERENCE  HANDB 0  OK 
OF  DISEASES  OF  THE  SKIN.  New  (3d)  edition.  12mo.  volume  of  637  pages, 
with  75  engravings,  and  one  colored  plate.    Cloth,  $2.50,  net. 

JAMIESON  (W.  ALLAN).  DISEASES  OF  THE  SKIN.  Third  edition.  Octavo, 
656  pages,  with  1  engraving  and  9  double-page  chromo-lithographic  plates.     Cloth,  $6. 

JEWETT  (CHARLES).  ESSENTIALS  OF  OBSTETRICS.  In  one  12mo.  volume 
of  356  pages,  with  80  engravings  and  3  colored  plates.     Cloth,  $2.25. 


THE  PRACTICE  OF  OBSTETRICS.    By  American  Authors.    One  large  octavo 

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JONES  'C.  HANDFIELD).  CLINICAL  OBSERVATIONS  ON  FUNCTIONAL 
NER  VOCS  DISORDERS.  Second  American  edition.  In  one  octavo  volume  of  340 
pages,     (loth,  $3.25. 

JULER  HENRY).  A  HANDBOOK  OF  OPHTHALMIC  SCIENCE  AND 
PEACTIGE.  Second  edition.  In  one  octavo  volume  of  549  pages,  with  201  engrav- 
ings, 17  chromo-lithographic  plates,  test-types  of  Jaeger  and  Snellen,  ami  Holmgren's 
Color-Blindneee  Test.     Cloth,  $5.50;  leather,  $6.50. 

KIRK /EDWARD  C).  OPERATIVE  DENTISTRY.  New  (2d)  Edition.  See 
American  Text-books  of  Dentistry,  page  2. 

KING  (A.  F.  Aj.     A   MANUAL   OF  OBSTETRICS.    New  (8th)  edition.     In  one 

L2mO.   volume  of  612  pages,  with  20  1  illustrations.      Cloth,  S2. ■".<>,  net. 

KLEIN    E.  .     ELEMENTS  OF  HISTOLOGY.     New  (5th)  edition.     Enonepockefr 
size  l2mo.  volume  of  506  pages,  with  296  engravings.     Cloth,  $2.00,  net. 
of  Manuals,  page  14. 


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LANDIS  (HENRY  G.).  THE  MANAGEMENT  OF  LABOR.  In  one  handsome 
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LA    ROCHE     (R.).     YELLOW   FEVER.     In    two   8vo.    volumes  of    1468  pages. 

Cloth,  $7. 

LAURENCE  (J.  Z.)  AND  MOON  (ROBERT  C).  A  HANDY-BOOK  OF 
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with  66  engravings.     Cloth,  $2.75. 

LEA  (HENRY  C.).  CHAPTERS  FROM  THE  RELIGIOUS  HISTORY  OF 
SPAIN;  CENSORSHIP  OF  THE  PRESS;  MYSTICS  AND  ILLUMINATI; 
THE  ENDEMONIADAS ;  EL  SANTO  NINO  DE  LA  GUARDIA ;  BRI- 
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A  HISTORY  OF  AURICULAR  CONFESSION  AND  INDULGENCES 

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FORMULARY   OF    THE   PAPAL    PENITENTIARY.      In    one  octavo 


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STUDIES  IN  CHURCH  HISTORY.  The  Rise  of  the  Temporal  Power- 
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SUPERSTITION  AND  FORCE;  ESSAYS  ON  THE  WAGER  OF  LAWt 


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AN  HISTORICAL  SKETCH  OF  SACERDOTAL  CELIBACY  IN  THE 


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pages.     Cloth,  $4.50. 

LOOMIS  (ALFRED  L.)  AND  THOMPSON  (W.  GILMAN),  Editors.  A  SYS- 
TEM OF  PR  A  CTICAL  MEDICINE.  In  Contributions  by  Various  American  Authors. 
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LUFF  (ARTHUR  P.).  MANUAL  OF  CHEMISTRY,  for  the  use  of  Students  of 
Medicine.  In  one  12mo.  volume  of  522  pages,  with  36  engravings.  Cloth,  $2.  See 
Students'  Series  of  Manuals,  page  14. 

LYMAN  (HENRY  M.).  THE  PRACTICE  OF  MEDICINE.  In  one  very  hand- 
some octavo  volume  of  925  pages  with  170  engravings.     Cloth,  $4.75 ;  leather,  $5.75. 

LYONS  (ROBERT  D.).    A  TREATISE  ON  FEVER.    In  one  octavo  volume  of  362 

pages.     Cloth,  $2.25. 

MACKENZIE  (JOHN  NOLAND).  THE  DISEASES  OF  THE  NOSE  AND 
THROAT.     Octavo,  of  about  600  pages,  richly  illustrated.     Preparing. 

MAISCH  (JOHN  M.).  A  MANUAL  OF  ORGANIC  MATERIA  MEDICA. 
New  (7th)  edition,  thoroughly  revised  by  H.  C.  C  Maisch,  Ph.G.,  Ph.D.  In  one  very 
handsome  12mo.  of  512  pages,  with  285  engravings.     Cloth,  $2.50,  net. 

MALSBARY  (GEO.  E.).  A  POCKET  TEXT-BOOK  OF  THEORY  AND 
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Cloth,  $1.75,  net;  flexible  red  leather,  $2.25,  net. 

MANUALS.  See  Students'  Quiz  Series,  page  14,  Students'  Series  of  Manuals,  page  14,  and 
Series  of  Clinical  Manuals,  page  13. 

MARSH  (HOWARD).  DISEASES  OF  THE  JOINTS.  In  one  12mo.  volume  of 
468  pages,  with  64  engravings  and  a  colored  plate.  Cloth,  $2.  See  Series  of  Clinical 
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MARTIN  (EDWARD.)  SURGICAL  DIAGNOSIS.  One  12mo.  volume  of  400 
pages,  richly  illustrated.     Preparing. 

MARTIN  (WALTON)  AND  ROCKWELL  (W.  H.,  JR.).  A  POCKET  TEXT- 
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trations.    Just  ready.     Cloth,  $1.50,  net;  flexible  red  leather,  $2.00,  net. 

MAY  (C.  H.).  MANUAL  OF  THE  DISEASES  OF  WOMEN.  For  the  use  of 
Students  and  Practitioners.  Second  edition,  revised  by  L.  S.  Rau,  M.D.  In  one  12mo. 
volume  of  360  pages,  with  31  engravings.     Cloth,  $1.75. 

MEDICAL  NEWS  POCKET  FORMULARY.    See  page  1. 

MITCHELL  (JOHN  K.).  REMOTE  CONSEQUENCES  OF  INJURIES  OF 
NERVES  AND  THEIR  TREATMENT.  In  one  handsome  12mo.  volume  of  239 
pages,  with  12  illustrations.     Cloth  $1.75. 

MITCHELL  (S.  WEIR).  CLINICAL  LESSONS  ON  NERVOUS  DISEASES. 
In  one  very  handsome  12mo.  volume  of  299  pages,  with  17  engravings  and  2  colored  plates. 
Cloth,  $2.50. 

MORRIS  (MALCOLM).  DISEASES  OF  THE  SKIN.  New  (2d)  edition.  In  one 
12mo.  volume  of  601  pages,  with  10  chromo-lithographic  plates  and  26  engravings. 
Cloth,  $3.25,  net. 

MOLLER  (J.).  PRINCIPLES  OF  PHYSICS  AND  METEOROLOGY.  In  one 
large  8vo.  volume  of  623  pages,  with  538  engravings.     Cloth,  $4.50. 

MUSSER  (JOHN  H.).  A  PRACTICAL  TREATISE  ON  MEDICAL  DIAG- 
NOSIS, for  Students  and  Physicians.  New  (4th)  edition.  In  one  octavo  volume  of 
1104  pages,  with  250  engravings  and  49  full-page  colored  plates.  Just  Ready.  Cloth, 
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NATIONAL  DISPENSATORY.     See  Stille,  Maisch  &  Caspari,  page  14. 

NATIONAL    FORMULARY.      See  National  Dispensatory,  page  14. 

NATIONAL  MEDICAL  DICTIONARY.    See  Billings,  page  3. 

NETTLESHIP  (E.).  DISEASES  OF  THE  EYE.  New  (6th)  American  from  sixth 
English  edition.  Thoroughly  revised.  12mo.,  562  pages,  with  192  engravings,  5  colored 
plates,  test-types,  formulae  and  color-blindness  test.     Cloth,  $2.25,  net. 

NICHOLS  (JOHN  B.)  AND  VALE  (F.  P.).  A  POCKET  TEXT-BOOK  OF 
HISTOLOGY  AND  PATHOLOGY.  12mo.  of  459  pages,  with  213  illustrations. 
Just  ready.     Cloth,  $1.75,  net;  flexible  red  leather,  $2.25,  net. 

NORRIS  (WM.  F.)  AND  OLIVER  (CHAS.  A.).  TEXT-BOOK  OF  OPHTHAL- 
MOLOGY. In  one  octavo  volume  of  641  pages,  with  357  engravings  and  5  colored 
plates.     Cloth,  $5 ;  leather,  $6. 

OWEN  (EDMUND).  SURGICAL  DISEASES  OF  CHILDREN.  In  one  12mo. 
volume  of  525  pages,  with  85  engravings  and  4  colored  plates.  Cloth,  $2.  See  Series  of 
Clinical  Manuals,  page  13. 

PARK  (WILLIAM  H.).  BACTERIOLOG  Y  IN  MEDICINE  AND  SURGER  Y. 
12mo.  688  pages,  with  87  engravings  in  black  and  colors  and  2  colored  plates.  Just 
Ready.     Cloth,  $3.00,  net. 

PARK  (ROSWELL),  Editor.  A  TREA  TISE  ON  SURGER  Y,  by  American  Authors. 
I'or  Students  and  Practitioners  of  Surgery  and  Medicine.  New  condensed  edition. 
in  one  large  octavo  volume  of  1261  pages,  with  625  engravings  and  38  plates.  Just 
Beady.     Cloth,  net,  $6.00;  leather,  net,  $7.00. 

PARVIN  (THEOPHILUSj.  THE  SCIENCE  AND  ART  OF  OBSTETRICS. 
Third  edition  In  one  handsome  octavo  volume  of  677  pages,  with  267  engravings  and 
2  colored  plates.     Cloth,  $4.25;  leather,  $5  25. 


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12  LEA     BROTHERS    &     CO.' S    PUBLICATIONS. 

PEPPER'S  SYSTEM  OF  MEDICINE.    See  page  2. 

PEPPER  (A.  J.).  SURGICAL  PATHOLOGY.  In  one  12mo  volume  of  511  pages, 
with  81  engravings.     Cloth,  $2.     See  Students'  Series  of  Manuals,  page  14. 

PICK  (T.  PICKERING).  FRACTURES  AND  DISLOCATIONS.  In  one  12mo. 
volume  of  530  pages,  with  93  engravings.    Cloth,  $2.    See  Series  of  Cliniml  Manuals,  p.  13. 

PLAYFAIR  (W.  S.).  A  TREATISE  ON  THE  SCIENCE  AND  PRACTICE 
OF  MIDWIFERY.  New  (7th)  American  from  the  Ninth  English  edition.  In  one 
octavo  volume  of  700  pages,  with  207  engravings  and  7  full  page  plates.  Cloth,  $3.75, 
net;  leather,  $4.75,  net. 

THE  SYSTEMATIC  TREATMENT  OF  NERVE  PROSTRATION  AND 

HYSTERIA.    In  one  12mo.  volume  of  97  pages.     Cloth,  $1. 

POLITZER  (ADAM).  A  TEXT-BOOK  OF  THE  DISEASES  OF  THE  EAR 
AND  ADJACENT  ORGANS.  Second  American  from  the  third  German  edition. 
In  one  octavo  volume  of  748  pages,  with  330  original  engravings. 

POCKET  FORMULARY.     See  page  1. 

POCKET  TEXT-BOOKS  Cover  the  entire  domain  of  medicine  in  sixteen  volumes  of 
350  to  450  pages  each,  written  by  teachers  in  leading  American  medical  colleges. 
Issued  under  the  editorial  supervision  of  Bern  B.  Gaxlatjdet,  M.D. ,  of  the  College  of 
Physicians  and  Surgeons,  New  York.  Thoroughly  modern  and  authoritative,  concise 
and  clear,  amply  illustrated  with  engravings  and  plates,  handsomely  printed  and 
bound.  The  series  is  constituted  as  follows  :  Anatomy  (preparing),  Physiology  (ready), 
Chemistry  and  Physics  (ready),  Histology  and  Pathology  (ready),  Materia  Medica, 
Therapeutics,  Medical  Pharmacy,  Prescription  Writing  and  Medical  Latin  (ready), 
Practice  (ready),  Diagnosis  (shortly),  Nervous  and  Mental  Diseases  (ready),  Surgery 
(preparing),  Genito-Urinary  and  Venereal  Diseases  {preparing),  Skin  Diseases 
(preparing),  Eye,  Ear,  Nose  and  Throat  (ready),  Obstetrics  (ready},  Gynecology 
(ready),  Diseases  of  Children  (ready),  Bacteriology  and  Hvgiene  (shortly).  For  further 
details  see  under  respective  authors  in  this  catalogue.  Special  circular  free  on  appli- 
cation. 

POTTS  (CHAS.  S.).  A  POCKET  TEXT-BOOK  OF  NERVOUS  AND 
MENTAL  DISEASES.  12mo.  of  455  pages,  with  88  illustrations.  Just  ready. 
Cloth,  $1.75,  net;  flexible  red  leather,  $2.25,  net. 

PROGRESSIVE  MEDICINE.    See  page  1. 

PURDY  (CHARLES  W.).  BRIGHT' S  DISEASE  AND  ALLIED  AFFEC- 
TIONS OF  THE  KIDNEY.  In  one  octavo  volume  of  288  pages,  with  18  engrav- 
ings.    Cloth,  $2. 

PYE-SMITH  (PHILIP  H.).     DISEASES  OF  THE  SKIN.     In  one  12mo.  volume 

of  407  pages,  with  28  illustrations,  18  of  which  are  colored.     Cloth,  $2. 

QUIZ  SERIES.     See  Students'  Quiz  Series,  page  14. 

RALFE  (CHARLES  H.).  CLINICAL  CHEMISTRY.  In  one  12mo.  volume  of 
314  pages,  with  16  engravings.     Cloth,  $1.50.     See  Students'  Series  of  Manuals,  page  14. 

RAMSBOTHAM  (FRANCIS  H.).  THE  PRINCIPLES  AND  PRACTICE  OF 
OBSTETRIC  MEDICINE  AND  SURGERY.  Imperial  octavo,  of  640  pages, 
with  64  plates  and  numerous  engravings  in  the  text.     Leather,  $7. 

REMSEN    (IRA).     THE  PRINCIPLES  OF  THEORETICAL    CHEMISTRY. 

New  (5th)  edition,  thoroughly  revised.     In  one  12mo.  volume  of  326  pages.     Cloth,  $2. 

RICHARDSON   (BENJAMIN  WARD).    PREVENTIVE  MEDICINE.    In  one 

octavo  volume  of  729  pages.     Cloth,  $4 ;  leather,  $5. 

ROBERTS  (JOHN  B.).  THE  PRINCIPLES  AND  PRACTICE  OF  MODERN 
SURGERY.  New  (2d)  edition.  'In  one  octavo  volume  of  838  pages,  with  474 
engravings  and  8  plates.     Cloth,  $4.25,  net;  leather,  $5.25,  net. 

THE  COMPEND  OF  ANATOMY.     For  use  in  the  Dissecting  Boom  and  in 

preparing  for  Examinations.     In  one  16mo.  volume  of  196  pages.     Limp  cloth,  75  cents. 


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LEA     BROTHERS    &     CO.'S    PUBLICATIONS.  13 

ROSS  (JAMES).    A  HANDBOOK  OF  THE  DISEASES  OF  THE  NERVOUS 

SYSTEM.     In  one  handsome  octavo  volume  of  726  pages,  with  184  engravings.     Cloth, 
$4.50;  leather,  $5.50. 

SCHAFER  ( EDWARD  A. ) .  THE  ESSENTIALS  OF  HISTOL OGY,  DESCRIP- 
TIVE AND  PRACTICAL.  For  the  use  of  Students.  New  (5th)  edition.  In  one 
handsome  octavo  volume  of  350  pages,  with  325  illustrations.    Cloth,  $3,  net. 

A    COURSE    OF  PRACTICAL    HISTOLOGY.     Second  edition.     In    one 


12mo.  volume  of  307  pages,  with  59  engravings.     Cloth,  $2.25. 

SCHLEIF  (WM.).  A  POCKET  TEXT-BOOK  OF  MATERIA  MEDIC  A, 
THERAPEUTICS,  PRESCRIPTION  WRITING.  MEDICAL  LATIN  AND 
MEDICAL  PHARMACY.  12mo.  352  pages.  Just  Ready.  Cloth,  $1.50,  net; 
flexible  red  leather,  $2.00,  net. 

SCHMITZ  AND  ZUMPT'S  CLASSICAL  SERIES. 
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SCHMITZ' S  ELEMENTARY  LATIN  EXERCISES.     Cloth,  50  cents. 
SALL  UST.     Cloth,  60  cents ;  half  bound,  70  cents. 
NEPOS.     Cloth,  60  cents ;  half  bound,  70  cents. 
VIRGIL.     Cloth,  85  cents;  half  bound,  $1. 
CURTIUS.     Cloth,  80  cents  ;  half  bound,  90  cents. 

SCHOFIELD  (ALFRED  T.).  ELEMENTARY  PHYSIOLOGY  FOR  STU- 
DENTS. In  one  12mo.  volume  of  380  pages,  with  227  engravings  and  2  colored  plates. 
Cloth,  $2. 

SCHREIBER  (JOSEPH).  A  MANUAL  OF  TREATMENT  BY  MASSAGE 
AND  METHODICAL  MUSCLE  EXERCISE.  Translated  by  Walter  Mendel- 
son,  M.  D.,  of  New  York.  In  one  handsome  octavo  volume  of  274  pages,  with  117  fine 
engravings. 

SENN  (NICHOLAS).  SURGICAL  BACTERIOLOGY.  Second  edition.  In  one 
octavo  volume  of  268  pages,  with  13  plates,  10  of  which  are  colored,  and  9  engravings. 
Cloth,  $2. 

SERIES  OF  CLINICAL  MANUALS.  A  Series  of  Authoritative  Monographs  on 
Important  Clinical  Subjects,  in  12mo.  volumes  of  about  550  pages,  well  illustrated.  The 
following  volumes  are  now  ready:  Carter  and  Frost's  Ophthalmic  Surgery,  $2. 25 ; 
Marsh  on  Diseases  of  the  Joints,  $2;  Owen  on  Surgical  Diseases  of  Children,  $2; 
Pick  on  Fractures  and  Dislocations,  $2. 

For  separate  notices,  see  under  various  authors'  names. 

SERIES  OF  POCKET  TEXT-BOOKS.     See  page  12. 

SERIES  OF  STUDENTS'  MANUALS.    See  next  page. 

SIMON  (CHARLES  E.).  CLINICAL  DIAGNOSIS,  BY  MICROSCOPICAL 
AND  CHEMICAL  METHODS.  New  (3d)  and  revised  edition.  In  one  handsome 
octavo  volume  of  563  pages,  with  138  engravings  and  18  full-page  plates  in  colors. 
Just  Ready.     Cloth,  $3.50,  net. 

SIMON  (W.).  MANUAL  OF  CHEMISTRY.  A  Guide  to  Lectures  and  Laboratory 
Work  for  Ik-ginners  in  Chemistry.  A  Text-book  specially  adapted  for  Students  of  Phar- 
macy and  Medicine.  Sixth  edition.  In  one  8vo.  volume  of  536  pages,  with  46 
engraving  and  8  plates  showing  colors  of  64  tests.     Cloth,  $3.00,  net. 

SLADE  (D.  D.j.    DIPHTHERIA;  ITS  NATURE  AND  TREATMENT.     Second 

edition.      In  one  royal  12mo.  volume,  158  pages.     Cloth,  $1.25. 

SMITH  (EDWARD).  CONSUMPTION;  ITS  EARLY  AND  REMEDIABLE 
STA  QE8.      In  one  8vo.  volume  of  253  pages.     Cloth,  $2.25. 

SMITH  'J.  LEWIS).  A  TREATISE  ON  THE  DISEASES  OF  INFANCY 
AND  CHILDHOOD.  Eighth  edition,  thoroughly  revised  and  rewritten  and  greatly 
enlarged.  In  one  lar^e  8vo.  volume  of  983  pages,  with  273  illustrations  and  4  full- 
page  plates.     Cloth,  $4.50;  leather,  $5. 50. 


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14  LEA    BROTHERS    &    CO.' S    PUB  L/C  AT/0  N  S. 

SMITH  (STEPHEN).  OPERATIVE  SURGERY.  Second  and  thoroughly  revised 
edition.     In  one  octavo  vol.  of  892  pages,  with  1005  engravings.     Cloth,  $4 ;  leather,  $5. 

SOLLY    (S.    EDWIN).     A    HANDBOOK    OF  MEDICAL    CLIMATOLOGY. 

In  one  handsome  octavo  volume  of  462  pages,  with  engravings  and  11  full-page  plates, 
5  of  which  are  in  colors.     Cloth,  $4.00. 

STILLE  (ALFRED).  CHOLERA;  ITS  ORIGIN,  HISTORY,  CAUSATION, 
SYMPTOMS,  LESIONS,  PREVENTION  AND  TREATMENT.  In  one  12mo. 
volume  of  163  pages,  with  a  chart  showing  routes  of  previous  epidemics.     Cloth,  $1.25. 

THERAPEUTICS  AND  MATERIA  MEDIC  A.    Fourth  and  revised  edition. 


In  two  octavo  volumes,  containing  1936  pages.     Cloth,  $10 ;  leather,  $12. 

STILLE  (ALFRED),  MAISCH  (JOHN  M.)  AND  CASPARI  (CHAS.  JR.). 
THE  NATIONAL  DISPENSATORY:  Containing  the  Natural  History,  Chemistry, 
Pharmacy,  Actions  and  Uses  of  Medicines,  including  those  recognized  in  the  latest  Phar- 
macopoeias of  the  United  States,  Great  Britian  and  Germany,  with  numerous  references 
to  the  French  Codex.  Fifth  edition,  revised  and  enlarged  in  accordance  with  and  em- 
bracing the  new  U.  S.  Pharmacopoeia,  Seventh  Decennial  Revision.  With  Supplement 
containing  the  new  edition  of  the  National  Formulary.  In  one  magnificent  imperial 
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ready  reference  Thumb-letter  Index.     Cloth,  $7.75;  leather,  $8.50. 

STIMSON  (LEWIS  A.).  A  MANUAL  OF  OPERATIVE  SURGERY.  New 
(4th)  edition.  In  one  royal  12mo.  volume  of  581  pages,  with  293  engravings.  Cloth,  $3.00, 
net.     Just  Ready. 

A    TREATISE   ON  FRACTURES  AND  DISLOCATIONS.     New  (3d) 


Edition.     In  one  handsome  octavo  volume  of  842  pages,  with  336  engravings  and  32 
full-page  plates.     Just  Ready.     Cloth,  $5  ;  leather,  $6,  net. 

STUDENTS'  QUIZ  SERIES.  A  New  Series  of  Manuals  in  question  and  answer  for 
Students  and  Practitioners,  covering  the  essentials  of  medical  science.  Thirteen  volumes, 
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and  issued  at  a  low  price.  1.  Anatomy  (double  number);  2.  Physiology;  3.  Chemistry 
and  Physics ;  4.  Histology,  Pathology  and  Bacteriology ;  5.  Materia  Medica  and  Thera- 
peutics ;  6.  Practice  of  Medicine ;  7.  Surgery  (double  number) ;  8.  Genito-Urinary  and 
Venereal  Diseases ;  9.  Diseases  of  the  Skin ;  10.  Diseases  of  the  Eye,  Ear,  Throat  and 
Nose ;  11.  Obstetrics ;  12.  Gynecology ;  13.  Diseases  of  Children.  Price,  $1  each,  except 
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Full  specimen  circular  on  application  to  publishers. 

STUDENTS'  SERIES  OF  MANUALS.  A  Series  of  Fifteen  Manuals  by  Eminent 
Teachers  or  Examiners.  The  volumes  are  pocket-size  12mos.  of  from  300-540  pages,  pro- 
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announced:  Herman's  First  Lines  in  Midwifery,  $1.25;  Luff's  Manual  of  Chemistry, 
$2;  Brtjce's  Materia  Medica  and  Therapeutics  (sixth  edition),  $1.50,  net;  Gould's  Sur- 
gical Diagnosis,  $2;  Klein's  Elements  of  Histology  (5th  edition),  $2.00,  net;  Pepper's 
Surgical  Pathology,  $2;  Treves'  Surgical  Applied  Anatomy,  $2;  Ralfe's  Clinical 
Chemistry,  $1.50;  and  Clarke  and  Lockwood's  Dissector's  Manual,  $1.50. 
For  separate  notices,  see  under  various  authors'  names. 

STURGES  (OCTAVIUS).  AN  INTRODUCTION  TO  THE  STUDY  OF  CLIN- 
ICAL MEDICINE.     In  one  12mo.  volume.     Cloth,  $1.25. 

SUTTON  (JOHN  BLAND).  SURGICAL  DISEASES  OF  THE  OVARIES 
AND  FALLOPIAN  TUBES.  _  Including  Abdominal  Pregnancy.  In  one  12mo.  vol- 
ume of  513  pages,  with  119  engravings  and  5  colored  plates.     Cloth,  $3. 

TAIT  (L AWSON ' .  DISEASES  OF _  WOMEN  AND  ABD  OMINAL  S UR GER  Y. 
Vol.  I.  contains  554  pages,  62  engravings,  and  3  plates.     Cloth,  $3. 

TANNER  (THOMAS  HAWKES).  ON  THE  SIGNS  AND  DISEASES  OF 
PREGNANCY.  From  the  second  English  edition.  In  one  octavo  volume  of  490  pages, 
with  4  colored  plates  and  16  engravings.     Cloth,  $4.25. 


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TAYLOR  (ALFRED  S.).  MEDICAL  JURISPRUDENCE.  New  American 
from  the  twelfth  English  edition,  specially  revised  by  Clark  Bell,  Esq.,  of  the  N.  Y. 
Bar.  In  one  octavo  volume  of  831  pages,  with  54  engravings  and  8  full-page  plates. 
Cloth,  $4.50;  leather,  $5.50. 

ON   POISONS    IN    RELATION    TO    MEDICINE    AND    MEDICAL 

JURISPRUDENCE.     Third  American  from  the  third  London  edition.     In  one  8vo. 
volume  of  788  pages,  with  104  illustrations.     Cloth,  $5.50;  leather,  $6.50. 

TAYLOR  (ROBERT  W.).  GENITO-URINARY  AND  VENEREAL  DIS- 
EASES AND  SYPHILIS  New  (2d)  edition.  In  one  very  handsome  octavo  volume 
of  720  pages,  with  135  engravings  and  27  colored  plates.  Just  ready.  Cloth,  $5.00,  net; 
leather,  $6.00,  net. 

A  PRACTICAL  TREATISE  ON  SEXUAL  DISORDERS  IN  THE  MALE 

AND  FEMALE.     New   (2d)   edition.     In  one  octavo  volume  of  434  pages,  with  91 
engravings  and  13  plates.     Just  Ready.     Cloth,  $3.00,  net. 

A    CLINICAL    ATLAS    OF    VENEREAL    AND    SKIN    DISEASES. 


Including  Diagnosis,  Prognosis  and  Treatment.  In  eight  large  folio  parts,  measuring 
14  x  18  inches,  and  comprising  213  beautiful  figures  on  58  full-page  chromo-lithographic 
plates,  85  fine  engravings,  and  425  pages  of  text.  Complete  work  now  ready.  Price  per 
part,  sewed  in  heavy  embossed  paper,  $2.50.  Bound  in  one  volume,  half  Russia,  $27  ; 
half  Turkey  Morocco,  $28.  For  sale  by  subscription  only.  Address  the  publishers.  Spec- 
imen plates  by  mail  on  receipt  of  10  cents. 

TAYLOR  (SEYMOUR).  INDEX  OF  MEDICINE.  A  Manual  for  the  use  of  Senior 
Students  and  others.    In  one  large  12mo.  volume  of  802  pages.     Cloth,  $3-75. 

THOMAS  (T.  GAILLARD)  AND  MUNDE  (PAUL  P.).  A  PRACTICAL 
TREATISE  ON  THE  DISEASES  OF  WOMEN.  Sixth  edition,  thoroughly 
revised  by  Paul  F.  Mtjnde,  M.D.  In  one  handsome  octavo  volume  of  824  pages,  with 
347  engravings.     Cloth,  $5  ;  leather,  $6. 

THOMPSON  (W.  GILMAN).    A    TEXT-BOOK  OF  PRACTICAL  MEDICINE. 

For  Students  and  Practitioners.  In  one  handsome  octavo  volume  of  1012  pages,  with 
79  illustrations.     Just  Ready.     Cloth,  $5.00,  net;  leather,  $6.00,  net. 

THOMPSON  (SIR  HENRY).  CLINICAL  LECTURES  ON  DISEASES  OF 
THE  URINARY  ORGANS.  Second  and  revised  edition.  In  one  octavo  volume  of 
203  pages,  with  25  engravings.     Cloth,  $2.25. 

THE  PATHOLOGY  AND   TREATMENT  OF  STRICTURE  OF  THE 

URETHRA  AND  URINARY  FISTULA.  From  the  third  English  edition.  In 
one  octavo  volume  of  359  pages,  with  47  engravings  and  3  lithographic  plates.  Cloth, 
$3.50. 

THOMSON  ( JOHN).  A  GUIDE  TO  THE  CLINICAL  EXAMINATION  AND 
TREA  TMENT  OF  SICK  CHILDREN.  In  one  crown  octavo  volume  of  350  pages 
with  52  illustrations.     Cloth,  $1.75,  net. 

TIRARD  NESTOR).  MEDICAL  TREATMENT  OF  DISEASES  AND  SYMP- 
TOMS.    J landsi mie  octavo  volume  of  627  pages.     Just  Ready.     Cloth,  $4.00,  net. 

TODD  ^ROBERT  BENTLEY).  CLINICAL  LECTURES  ON  CERTAIN 
ACUTE  DISEASES.     In  one  8vo.  volume  of  320  pages.    Cloth,  $2.50. 

TREVES  'FREDERICK).  OPERATIVE  SURGERY.  In  two  8vo.  volumes  con- 
taining L560  p&g6B,  with  422  illustrations.     Cloth,  $9;  leather,  $11. 


A  SYSTEM  OF  8UBOEET.  In  Contributions  by  Twenty-five  English  Sur- 
geons. In  two  large  octavo  volumes,  containing  2298  pages,  with  950  engravings  and 
4  full-page  plates.     Per  volume,  cloth,  $8. 

SURGICAL    APPLIED    ANATOMY.     In  one  12mo.   volume  of  583  pages, 


with  <i]  engravings.     Cloth,  $2.     Sec  Sluilmi:'  Series  of  Manuals,  page  14. 

TUTTLE  GEO.  M.).  A  POCKET  TEXT-BOOK  OF  DISEASES  OF 
CHILDREN.  l2mo.  874  pages,  with  5  plates.  Just  Ready.  Cloth,  $1.50,  net; 
flexible  red  leather,  $2.00,  net. 


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16  LEA    BROTHERS    &     CO.' S    PUBLICATIONS. 

VAUGHAN  (VICTOR  0.)  AND  NOVY  (FREDERICK  G.).  PTOMAINS, 
LEUCO MAINS,  TOXINS  AND  ANTITOXINS,  or  the  Chemical  Factors  in  the 
Causation  of  Disease.     Third  edition.     In  one  12mo.  volume  of  603  pages. 

VISITING  LIST.  THE  MEDICAL  NEWS  VISITING  LIST  for  1901.  Four 
styles  :  Weekly  (dated  for  30  patients) ;  Monthly  (undated  for  120  patients  per  month) ; 
Perpetual  (undated  for  30  patients  each  week) ;  and  Perpetual  (undated  for  60  patients 
each  week).  The  60-patient  book  consists  of  256  pages  of  assorted  blanks.  The  first 
three  styles  contain  32  pages  of  important  data,  thoroughly  revised,  and  160  pages  of 
assorted  blanks.  Each  in  one  volume,  price,  $1.25.  With  thumb-letter  index  for  quick 
use,  25  cents  extra.  Special  rates  to  advance-paying  subscribers  to  The  Medical  News 
or  The  American  Journal  of  the  Medical  Sciences,  or  both,    See  page  1. 

WATSON  (THOMAS).  LEC TUBES  ON  THE  PRINCIPLES  AND  PRAC- 
TICE OF  PHYSIC.  American  edition  from  the  fifth  and  enlarged  English,  with 
additions  by  H.  Hartshorne,  M.D.  In  two  large  8vo.  volumes  of  1840  pages,  with  190 
engravings.     Cloth,  $9 ;  leather,  $11. 

WEST  (CHARLES).  LECTURES  ON  THE  DISEASES  PECULIAR  TO 
WOMEN.  Third  American  from  the  third  English  edition.  In  one  octavo  volume  of 
543  pages.     Cloth,  $3.75;  leather,  $4.75. 

ON  SOME  DISORDERS  OF  THE  NERVOUS  SYSTEM  IN  CHILD- 


HOOD.    In  one  small  12mo.  volume  of  127  pages.     Cloth,  $1. 

WHARTON  (HENRY  R.).  MINOR  SURGERY  AND  BANDAGING.  New 
(4th)  edition.  In  one  12mo.  volume  of  596  pages,  with  502  engravings,  many  of  which 
are  photographic.     Cloth,  $3.00,  net. 

WHITMAN  (ROYAL).  ORTHOPEDIC  SURGERY.  One  octavo  volume  of 
about  650  pages,  with  about  400  illustrations.     Preparing. 

WHITLA  (WILLIAM).  DICTIONARY  OF  TREATMENT,  OR  THERA- 
PEUTIC INDEX.  Including  Medical  and  Surgical  Therapeutics.  In  one  square 
octavo  volume  of  917  pages.     Cloth,  $4. 

WILLIAMS  (DAWSON).  MEDICAL  DISEASES  OF  INFANCY  AND 
CHILDHOOD.  New  (2d)  edition,  specially  revised  for  America  by  F.  S.  Churchill, 
A.M.,  M.D.  In  one  octavo  volume  of  53 3  pages,  with  52  illustrations  and  2  colored 
plates.     Cloth,  $3.50,  net.     Just  ready. 

WILSON  (ERASMUS).  A  SYSTEM  OF  HUMAN  ANATOMY.  A  new  and 
revised  American  from  the  last  English  edition.  Illustrated  with  397  engravings.  In 
one  octavo  volume  of  616  pages.     Cloth,  $4 ;  leather,  $5. 

WINCKEL  ON  PATHOLOGY  AND  TREATMENT  OF  CHILDBED.  In  one 
octavo  volume  of  484  pages.     Cloth,  $4. 

WOHLER'S  OUTLINES  OF  ORGANIC  CHEMISTRY.  Translated  from  the 
eighth  German  edition,  by  Ira  Remsen,  M.D.  In  one  12mo.  volume  of  550  pages. 
Cloth,  $3. 

YEARBOOK  OF  TREATMENT  FOR  1898.  A  Critical  Review  for  Practitioners  of 
Medicine  and  Surgery.  In  contributions  by  24  well-known  medical  writers.  12mo.,  488 
pages.     Cloth,  $1.50 

YEAR-BOOKS  OF  TREATMENT  for  1892,  1893,  1896,  and  1897,  similar  to  above. 
Each,  cloth,  $1.50. 

YOUNG  (JAMES  K.).  ORTHOPEDIC  SURGERY.  In  one  8vo  volume  of  475 
pages,  with  286  illustrations.     Cloth,  $4 ;  leather,  $5. 


Philadelphia,  706,  708  and  710  Sansom  St— New  York,  111  Fifth  Avenue. 


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